Fusion-intermediate state of HIV-1 gp41 targeted by broadly neutralizing antibodies

ABSTRACT

Isolated, antigenic polypeptides including a prehairpin intermediate conformation of gp41 and vectors encoding such polypeptides are provided. Antibodies that bind to a prehairpin intermediate conformation of gp41 and methods of making antibodies a that bind to prehairpin intermediate conformation of gp41 are also provided. Vaccines against a prehairpin intermediate conformation of gp41, as well as methods of treating subjects infected with HIV, preventing HIV infection, and inhibiting HIV-mediated activities are also provided. Methods of screening compounds that bind to an isolated, prehairpin intermediate conformation of gp41 are further provided.

RELATED APPLICATIONS

This application is a continuation of PCT/US2009/035459, filed Feb. 27,2009 which claims priority from U.S. provisional patent application No.61/032,732, filed Feb. 29, 2008, and U.S. provisional patent applicationNo. 61/032,520, filed Feb. 29, 2008, each of which is herebyincorporated herein by reference in its entirety for all purposes.

STATEMENT OF GOVERNMENT INTERESTS

This invention was made with government support under NationalInstitutes of Health grant numbers R21 AI069972 and U01 AI067854. TheGovernment has certain rights in the invention.

FIELD

The present invention relates to methods and compositions forneutralizing viral infection, for example, infection by HIV (e.g.,HIV-1).

BACKGROUND

HIV-1 infection generally induces a strong antibody response to theenvelope glycoprotein (i.e., trimeric (gp160)₃, cleaved to(gp120/gp41)₃), the sole antigen on the virion surface. Most of theinduced antibodies are ineffective in preventing infection, however, asthey are either non-neutralizing or narrowly isolate-specific, and thevirus replicates so rapidly that ongoing selection ofneutralization-resistant mutants allows viral evolution to “keep ahead”of high-affinity antibody production (Wei et al. (2003) Nature 422:307).Moreover, much of the antibody response may be to rearranged ordissociated forms of gp120 and gp41, on which the dominant epitopes maybe either in hypervariable loops or in positions occluded onvirion-borne envelope trimer. A few rare “broadly neutralizing”antibodies have been detected that recognize one of three relativelyconserved regions on the envelope protein: the CD4

binding site (mAb b12) (Burton et al. (1994) Science 266:1024);carbohydrates on the outer gp120 surface (mAb 2G12) (Trkola et al.(1996) J. Virol. 70:1100); and a segment of the gp41 ectodomain adjacentto the viral membrane (mAbs 2F5 and 4E10) (Muster et al. (1993) J.Virol. 67:6642; Stiegler et al. (2001) AIDS Res. Hum. Retroviruses17:1757), often called the membrane-proximal external region (“MPER”).Understanding the molecular mechanisms of neutralization by these andother antibodies could help design immunogens to induce them.

Fusion of viral and target cell membranes initiates HIV-1 infection.Conformational changes in gp120 that accompany its binding to receptor(CD4) and co-receptor (e.g., CCR5 or CXCR4) lead to dissociation ofgp120 from gp41 and a cascade of refolding events in the latter(Harrison (2005) Adv. Virus Res. 64:231). In the course of theserearrangements, the N-terminal “fusion peptide” of gp41 translocates andinserts into the target cell membrane. A proposed extended conformationof the gp41 ectodomain, with its fusion peptide thus inserted and thetransmembrane anchor still in the viral membrane, has been called the“prehairpin intermediate” (Chan and Kim (1998) Cell 93:681). Theprehairpin intermediate is the target of various fusion inhibitors,including T-20/Enfuvirtide, the first approved fusion-inhibitingantiviral drug (Kilby and Eron (2003) N. Engl. J. Med. 348:2228). Thecharacteristics of the intermediate have been deduced from theproperties of these inhibitors or mimicries by short gp41 fragments(Eckert et al. Cell 99:103; Root et al. (2001) Science 291:884).Subsequent rearrangements from the intermediate to the postfusion stateof gp41 involve folding back of each of the three chains into ahairpin-like conformation, with two anti-parallel α-helices connected bya disulfide-containing loop. This process brings the fusion peptide andtransmembrane anchor, and hence the two membranes, close together at thesame end of the refolded protein.

SUMMARY

The present invention is directed in part to the discovery of where, inthe sequence of HIV envelope-mediated fusion events, neutralizingantibodies intervene and whether any such antibodies neutralize morethan a narrow range of isolates. Biochemically homogeneous forms of theHIV envelope glycoprotein were prepared having defined and uniformantigenic properties. These forms included at least one of each of theprincipal conformational states of the gp41 ectodomain: the prefusionconformation, the prehairpin intermediate, and the postfusionconformation. The present invention evidences that the epitopes for theMPER antibodies 2F5 and 4E10 are exposed only on a form of the envelopeprotein designed to mimic an intermediate in the transition from the“prefusion” conformation of the envelope, as found on infectiousvirions, to the “postfusion” conformation, the final, stable state ofgp41 after entry is complete. These results help explain the rarity of2F5- and 4E10-like antibody responses and indicate how one of skill inthe art can design immunogens to elicit them.

Accordingly, in certain exemplary embodiments, an isolated, antigenicpolypeptide comprising a prehairpin intermediate conformation of gp41 isprovided. The polypeptide includes a first heptad repeat 2 motif, asecond heptad repeat 2 motif, a heptad repeat 1 motif, and amembrane-proximal external region. The polypeptide can elicit productionof a broadly neutralizing antibody against HIV when injected into asubject. In certain aspects, the polypeptide includes one or more of thefollowing: a C-C loop domain between the second heptad repeat 2 motifand the membrane-proximal external region; a linker between the firstheptad repeat 2 motif and the heptad repeat 1 motif; an oligomerizationdomain (e.g., a trimerization domain) carboxy terminal to themembrane-proximal external region; and a protein tag carboxy terminal tothe membrane-proximal external region.

In certain exemplary embodiments, an isolated, antigenic polypeptidecomprising a prehairpin intermediate conformation of gp41 is providedhaving the following order, listed from the amino terminus to thecarboxy terminus: a first heptad repeat 2 motif; a heptad repeat 1motif; a C-C loop domain; a second heptad repeat 2 motif; and amembrane-proximal external region. In certain aspects, the polypeptideelicits production of a broadly neutralizing antibody when injected intoa subject.

In certain exemplary embodiments, a vector expressing a polynucleotideencoding a polypeptide comprising a prehairpin intermediate conformationof gp41 is provided. The vector includes the following motifs anddomains in the following order (listed from the amino terminus to thecarboxy terminus): a first heptad repeat 2 motif; a heptad repeat 1motif; a C-C loop domain; a second heptad repeat 2 motif; and amembrane-proximal external region.

In certain exemplary embodiments, a method of therapeutically treating asubject infected with HIV is provided. The method includes contacting asubject infected with HIV with a polypeptide comprising an isolated,prehairpin intermediate conformation of gp41 including a first heptadrepeat 2 motif, a second heptad repeat 2 motif, a heptad repeat 1 motifand a membrane-proximal external region, and eliciting an immuneresponse in the subject to therapeutically treat the subject. In certainaspects, a broadly neutralizing antibody is produced in the subject. Incertain aspects, the HIV titer in the subject infected with HIV isdecreased. In other aspects, the HIV is HIV-1. In yet other aspects, HIVinfection is eliminated from the HIV-infected subject.

In certain exemplary embodiments, a method of inhibiting an HIV-mediatedactivity in a subject in need thereof is provided. The method includescontacting an HIV-infected subject with a polypeptide comprising anisolated, prehairpin intermediate conformation of an envelopeglycoprotein including a first heptad repeat 2 motif, a second heptadrepeat 2 motif, a heptad repeat 1 motif and a membrane-proximal externalregion to inhibit the HIV-mediated activity. In certain aspects, theHIV-mediated activity is viral spread. In other aspects, HIV titer inthe HIV-infected subject is decreased.

In certain exemplary embodiments, a method of preventing HIV infectionin a subject including contacting a subject with a polypeptidecomprising an isolated, prehairpin intermediate conformation of gp41including a first heptad repeat 2 motif, a second heptad repeat 2 motif,a heptad repeat 1 motif and a membrane-proximal external region, andeliciting an immune response against the polypeptide in the subject isprovided. In certain aspects, a broadly neutralizing antibody againstHIV is raised in the subject.

In certain exemplary embodiments, a method of screening a compound thatbinds to an isolated, prehairpin intermediate conformation of gp41including providing a polypeptide including an isolated, prehairpinintermediate conformation of gp41 having a first heptad repeat 2 motif,a second heptad repeat 2 motif, a heptad repeat 1 motif and amembrane-proximal external region, contacting the polypeptide with thecompound, and determining the ability of the compound to bind to thepolypeptide is provided. In certain aspects, the compound inhibits anHIV-mediated activity. In other aspects, the compound is provided in alibrary.

In certain exemplary embodiments, a vaccine having an epitope comprisingan isolated, prehairpin intermediate conformation of gp41 including afirst heptad repeat 2 motif, a second heptad repeat 2 motif, a heptadrepeat 1 motif and a membrane-proximal external region is provided.

In certain exemplary embodiments, an anti-gp41 antibody specific againstan epitope comprising an isolated, prehairpin intermediate conformationof gp41 including a first heptad repeat 2 motif, a second heptad repeat2 motif, a heptad repeat 1 motif and a membrane-proximal external regionis provided.

In certain exemplary embodiments, a method of making an anti-gp41antibody comprising the steps of providing a host, contacting the hostwith an epitope comprising an isolated, prehairpin intermediateconformation of gp41 including a first heptad repeat 2 motif, a secondheptad repeat 2 motif, a heptad repeat 1 motif and a membrane-proximalexternal region, and allowing production of an anti-gp41 antibody in thehost is provided. In certain aspects, polyclonal antibodies are isolatedfrom the host. In other aspects, a lymphocyte is isolated from the host,and, optionally, a monoclonal antibody is made from the lymphocyte.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee. The foregoing and other features and advantages ofthe present invention will be more fully understood from the followingdetailed description of illustrative embodiments taken in conjunctionwith the accompanying drawings in which:

FIG. 1 schematically depicts the design of expression constructs ofHIV-1 envelope glycoproteins. Gp160 is the full-length precursor.Various segments of gp120 and gp41 are designated as follows: C1-C5,conserved regions 1-5; V1-V5, variable regions 1-5; F, fusion peptide;HR1, heptad repeat 1; C-C loop, the immunodominant loop with a conserveddisulfide bond; HR2, heptad repeat 2; MPER, membrane proximal externalregion (see FIG. 9); TM, transmembrane anchor; CT, cytoplasmic tail.Expression constructs are: gp140, the uncleaved ectodomain of gp160 witha His-tag at its C-terminus; gp140-Fd, the uncleaved ectodomain of gp160with a trimerization tag and a His-tag at its C-terminus; gp41-inter,gp41 in the prehairpin intermediate conformation trapped by anN-terminal HR2 peptide- and a C-terminal foldon tag; gp41-post, gp41 inthe six helix conformation with partial MPER representing the postfusionstate. Glycans are represented by tree-like symbols. At the bottom,diagrams represent the three dimensional organization of these proteinspecies. Gp120 and gp41 in the prefusion state are shown in light greenand light blue, respectively. The viral membrane is in orange. Otherregions are colored as in the schematics above.

FIGS. 2A-2F depict protein preparations of the prefusion and thepre-hairpin intermediate conformations of HIV-1 gp41. (A) Purified HIV-192UG-gp140-Fd trimer was resolved by gel-filtration chromatography usinga Superose 6 column. The apparent molecular mass was calculated based ona standard curve using the following known standards: thyoglobulin (670kDa), ferritin (440 kDa) and catalase (232 kDa). Peak fractions werepooled and analyzed by Coomassie stained SDS-PAGE (inset). (B)Analytical ultracentrifugation for 92UG-gp140-Fd trimer was performed ona Beckman XL-A analytical ultracentrifuge at 4° C. Three proteinconcentrations (0.62, 1.24, 2.48 μM) and three rotor speeds (5000, 7000,9000 rpm) were used. The data shown were collected with the protein at1.24 μM and rotor speed of 7000 rpm. Data sets were fitted to a singlespecies model and the protein partial specific volume was calculated tobe 0.686 ml/g based on the sugar content (Laue et al. (1992) inAnalytical Ultracentrifugation in Biochemistry and Polymer Science, ed.S. E. Harding AJRaJCH (Royal Society of Chemistry, Cambridge), pp.90-125). The molecular mass determined was 409+/−10 kDa. (C)92UG-gp140-Fd trimer was treated with various concentrations (lanes 1 to7, 0, 0.05, 0.25, 0.5, 1, 2, 5 mM, respectively) of EGS (ethylene glycolbis(succinimidylsuccinate)). The crosslinked products were analyzed bySDS-PAGE in a 4% gel. The molecular weight standard was cross-linkedphosphorylase b (Sigma). The dimeric and trimeric species of92UG-gp140-Fd migrate faster than expected for their molecular weights,probably due to their compactness after cross-linking (D)92UG-gp41-inter was expressed from E. coli and refolded in vitro. Therefolded protein was resolved by gel-filtration chromatography onSuperdex 200. The protein migrated on SDS-PAGE as a band of 26 kDa whensample was boiled and reduced. When not boiled and not reduced, theprotein migrated as a ladder of three bands of 26, 50, 80 kDa,respectively, corresponding to monomer, dimer and trimer (shown ininset, lane 1 and 2). (E) Analytical ultracentrifugation for92UG-gp41-inter was performed on a Beckman XL-A analyticalultracentrifuge at 4° C. Three protein concentrations (0.98, 1.96, 3.92μM) and three rotor speeds (15000, 17500, 26000 rpm) were used. The datashown were collected with the protein at 3.92 μM and rotor speed of17500 rpm. Data sets were fitted to a single species model and themolecular weight determined was 92+/−6 kDa. (F) 92UG-gp41-inter examinedby negative-stain electron microscopy. A view of a raw image is shown.The bottom row shows selected images after class averaging to increasethe signal-to-noise ratio. The dimensions of the rod-like molecules areroughly 150 Å×45 Å. The bar represents 20 nm.

FIGS. 3A-3D depict evidence that the broadly neutralizing mAb 2F5targets the prehairpin intermediate conformation. (A) 2F5 Fab wasimmobilized on a CM-5 chip, and 92UG-gp140-Fd (1 μM) or 92UG-gp140(without Foldon tag, 1 μM) were passed over the surface. The sensorgramfor 92UG-gp140-Fd is shown in pink, 92UG-gp140-Fd at 37° C. in orangeand 92UG-gp140 in red. The 92UG-gp140-Fd was cleaved by human plasmin,and the cleaved gp140-Fd was further purified away from aggregatedprotein and plasmin by gel-filtration chromatography on a prep-gradeSuperdex 200 column. The peak fraction containing cleaved, trimericgp140-Fd was immobilized on a Ni-NTA chip, and 2F5 Fab at 1 μM waspassed over the surface. The sensorgram for partially cleaved gp140-Fdis shown in black. No binding of 2F5 to any of the gp140 proteins wasobserved. As shown in the inset, 2F5 did react on an immunoblot with92UG-gp140-Fd (lane 1), as well as the two gp41 proteins in theprehairpin intermediate conformation, 92UG-gp41-inter-Fd (lane 2) andHXB2-gp41-inter-GCN4 (lane 3). (B) The Fab fragment of mAb 2F5 wasimmobilized on a CM-5 chip. Solutions at various concentrations of92UG-gp41-inter-Fd, the gp41-inter protein derived from the 92UG037.8sequence with a foldon tag were passed over the chip surface. Bindingkinetics were evaluated using BiaEvaluation software (Biocore) and a 1:1Langmuir binding model. The recorded sensorgrams are shown in black andthe fits in green. (C) The Fab fragment of 2F5 was immobilized on a CM-5chip. Solutions of 92UG-gp41-post at various concentrations (1.0, 2.5,5.0 and 10.0 μM) were passed over the chip surface. The recordedsensorgrams are shown in black for 92UG-gp41-post and in green for thefits. (D) T20 peptide at different concentrations (5, 10, 25 and 50 nM)was passed over a surface of a chip bearing immobilized 2F5 Fab. Therecorded sensorgrams are shown in black and the fits in green. Allinjections were carried out in duplicate and gave essentially the sameresults. Only one of the duplicates is shown in the figure.

FIGS. 4A-4D depict that the mAb 4E10 also targets the prehairpinintermediate conformation of gp41. (A) To preserve its nativeconformation, 92UG-gp140-Fd trimer was immobilized on a Ni-NTA chip, and4E10 IgG, 4E10 Fab and 240-D IgG, all at 1 μM, were passed over thesurface sequentially. Regeneration was not necessary after binding by4E10 IgG and Fab. The recorded sensorgrams are shown in blue for 240-D,in pink for 4E10 IgG and in red for 4E10 Fab. The IgG of 4E10 showedonly very weak binding, even with a potential avidity effect. (B) The92UG-gp41-inter protein was immobilized on a CM-5 chip. A solution of4E10 scFv at 50 nM was passed over the chip surface. Second injection ofa duplicate run gave lower binding level because harsh conditions had tobe used to regenerate the chip surface and led to lower baseline level.The experiment was repeated using a different chip and gave the similarresult. The recorded sensorgrams are shown in black for 4E10 scFv and ingreen for the fit. (C) Western blot of 92UG-gp140-Fd trimer and92UG-gp41-inter detected by mAb 4E10. Both proteins reacted with mAb4E10, as shown in lane 1 for 92UG-gp140-Fd, and lane 2 for92UG-gp41-inter (lane 2). (D) Solutions of 4E10 scFv at variousconcentrations (25-500 nM) were passed over the surface of an SA chipbearing immobilized biotinylated 4E10 epitope peptide. The recordedsensorgrams are shown in black and the fits in green. All injectionswere carried out in duplicate and gave essentially the same resultsexcept in B. Only one of the duplicates is shown in the figure.

FIG. 5 depicts expression of HIV-1 gp140 in insect cells. HIV-1 gp140from a series of isolates were expressed in two formats, with or withouta foldon trimerization tag at the C-terminus (e.g. gp140 andgp140-Foldon). The HIV-1 strains shown here include Bcon, the consensussequence of Glade B; Ccon, the consensus sequence of Glade C; 96ZM651.8,a Glade B sequence from chronic infection; 92UG037.8, a Glade Asequence; C97ZA012, a Glade C sequence; 02ZM.233M.6, a Glade C sequencefrom early infection; 01US.TRJO4551.58, a Glade B sequence from acuteinfection; and BCF03, a group O sequence. Sf9 cells were infected withthe corresponding baculovirus vectors, and cell supernatants wereharvested three days post-infection. Each protein contained a sixhistidine tag at its C-terminus. The secreted gp140 protein was analyzedby SDS-PAGE and immunoblotting with an anti-His-tag antibody. There wasno obvious correlation for secretion level, which, without intending tobe bound by scientific theory, presumably corresponds to proteinstability.

FIGS. 6A-6I depict characterization of the 92UGgp140-Fd trimer. (A) and(B) 92UGgp140-Fd trimer was immobilized on a CM-5 chip, and solublefour-domain CD4 (in A) and the Fab fragment of broadly neutralizingantibody 2G12 (in B) at various concentrations were passed over the chipsurface. Binding kinetics was evaluated using BiaEvaluation software(Biacore) using a two-step binding model for CD4 and a 1:1 Langmuirbinding model for 2G12. The recorded sensorgrams are shown in black andthe fits in green. Binding constants are summarized in Table 1. (C)Monomeric 92UG-gp120 core protein at 1 μM and trimeric 92UG-gp140-Fdprotein at 1 μM, respectively, were flowed over a CM-5 chip surfacecoated with the whole IgG of mAb b12. The recorded sensorgram for92UG-gp120 core is shown in black and the fit in green. The single curvewas fit to a 1:1 Langmuir binding model as published (Rits-Volloch etal. (2006) Embo J. 25:5026). The sensorgram of 92UGgp140-Fd trimer is inred. (D) and (E) Monomeric 92UG-gp120 core protein at 0.2 μM (in D) and1 μM (in E), and trimeric 92UG-gp140-Fd protein at 1 μM, respectively,were flowed over a CM-5 chip surfaces coated with the whole IgG of mAbb6 (in D) and mAb 15e (in E). The recorded sensorgram for 92UG-gp120core is shown in black and the fit in green. The single curve was fit toa 1:1 Langmuir binding model. The sensorgram of 92UGgp140-Fd trimer isin red. (F) Trimeric 92UG-gp140-Fd protein alone (0.5 μM), soluble CD4alone (5 μM) and a pre-incubated complex with 0.5 μM of 92UG-gp140-Fdprotein and 5 μM of soluble CD4 were passed over a mAb 17b surface on aCM-5 chip. The recorded sensorgram for CD4 alone is shown in black, for92UGgp140-Fd in blue and for the complex in red. (G) Two anti-gp41cluster I antibodies, 240-D or 246-D, were immobilized on a CM-5 chip,and 92UG-gp140-Fd trimer at 1 μM, was passed over the chip surface. Therecorded sensorgram for 240-D and 246-D are shown in black and in blue,respectively. All injections were carried out in duplicate and gaveessentially the same results. Only one of the duplicates is shown in thefigure. (H) Immobilization of 4E10 antibody to a CM5 chip directly bythe standard amine coupling procedure results in blocking binding to itsantigens. To generate a functional 4E10 surface to further confirmbinding results derived from using 4E10 antigens as immobilized ligands,purified protein A (Calbiochem) was immobilized to a CM5 chip first and4E10 IgG was allowed to bind to the surface. 92UGgp140-Fd and92UGgp41-inter at 1 μM, respectively, were passed over the surfacesequentially. No regeneration was needed after the 92UGgp140-Fd bindingstep. The recorded sensorgram for 92UGgp140-Fd is shown in red and for92UGgp41-inter in black. (I) The time-course of cleavage of the92UG-gp140-Fd protein by human plasmin (Sigma) was carried out at roomtemperature. The first lane on the left is gp140 alone; the last lane onthe right is human plasmin alone. Lane 2-9, cleavage proceeded for 5,10, 15, 20, 30, 60, 90 and 120 minutes. The N-termini of the cleavageproducts, gp120 and gp41, were confirmed by protein sequencing.

FIGS. 7A-7D depict the characterization of gp41-inter by surface plasmonresonance assay and circular dichroism. (A) Circular dichroism (CD)spectrum of 92UG-gp41-inter. The CD spectrum was recorded at 25° C. withprotein concentration of 0.71 μM in PBS. The spectrum was corrected withbaseline spectra recorded from buffer alone under the same conditions.(B) The Fab fragment of mAb 2F5 was immobilized on a CM-5 chip.Solutions at various concentrations of HXB2-gp41-inter-GCN4, thegp41-inter protein derived from the HXB2 sequence with a trimeric GCN4tag, were passed over the chip surface. Binding kinetics were evaluatedusing BiaEvaluation software (Biocore) and a 1:1 Langmuir binding model.The recorded sensorgrams are shown in black and the fits in green. (C)Two anti-gp41 cluster I antibodies, 240-D or 246-D were immobilized on aCM-5 chip, and, 92UG-gp41-inter-Fd at 1 μM, was passed over the chipsurface. The second injection of a duplicate run gave lower bindinglevel because harsh conditions had to be used to regenerate the chipsurface and led to lower baseline level. The recorded sensorgrams fromthe first injection for 240-D and 246-D are shown in black and in blue,respectively. (D) HXB2gp41-interf-GCN4 is not his-tagged and containsthe full epitope of 4E10. The his-tagged 4E10 scFv was immobilized toNi-NTA chip. HXB2gp41-interf-GCN4 at 50 nM was passed over the surface.The recorded sensorgram is shown in black and the fit in green. Allinjections were carried out in duplicate and gave essentially the sameresults except in (C). Only one of the duplicates is shown in thefigure. Binding constants are summarized in Table 1.

FIGS. 8A-8D depict that mAb 2F5 binds weakly to gp41 in its postfusionconformation. (A) 92UG-gp41-post was expressed from E. coli andextracted from cell pellets by an acid-extraction procedure (seeMethods). The protein was purified by HPLC on a C18 column (Vydac) andrefolded by a rapid-dilution protocol (see Methods). The refoldedprotein was concentrated and resolved by gel-filtration chromatographyon Superdex 200. The protein migrated on SDS-PAGE as a band of 11 kDa(shown in inset), as expected. (B) Analytical ultracentrifugation wasperformed on a Beckman XL-A analytical ultracentrifuge at 4° C. Threeprotein concentrations (0.98, 1.96, 3.92 μM) and three rotor speeds(23000, 28000, 39000 rpm) were used. The data shown were collected withthe protein at 3.92 μM and rotor speed of 23000 rpm. Date sets werefitted to a single species model and the molecular weight determined is34.1+/−0.6 kDa. (C) CD spectrum of 92UG-gp41-post. Circular dichroism(CD) spectrum was recorded at 25° C. with protein concentration of 0.56mg/ml in PBS. The spectrum was corrected with baseline spectra recordedfrom buffer alone under the same conditions. (D) The Fab fragment of thebroadly neutralizing antibody 2F5 was immobilized on a CM-5 chip.Solutions of 92UG-gp41-post at various concentrations (1.0, 2.5, 5.0 and10.0 μM) were passed over the chip surface. The recorded sensorgrams areshown in black for 92UG-gp41-post and in green for the fits.

FIG. 9 depicts a sequence alignment of the MPER regions of theconstructs used.

Sequences of the membrane-proximal external region from the variousconstructs and peptides are listed. The 2F5 epitope is highlighted inred and the 4E10 epitope in magenta. Sequence identifiers are asfollows: 92UGgp140-Fd is set forth as SEQ ID NO:1; HXB2gp41interf is setforth as SEQ ID NO:2; 92UGgp41inter is set forth as SEQ ID NO:3;92UGgp41post is set forth as SEQ ID NO:4; 2F5 epitope peptide is setforth as SEQ ID NO:5; and 4E10 epitope peptide is set forth as SEQ IDNO:6.

DETAILED DESCRIPTION

Most antibodies induced by HIV-1 are ineffective at preventinginitiation or spread of infection, as they are either non-neutralizingor narrowly isolate-specific. One of the biggest challenges in HIVvaccine development is to design an HIV envelope immunogen that caninduce protective, neutralizing antibodies effective against the diverseHIV-1 strains that characterize the global pandemic. Indeed, thegeneration of “broadly neutralizing” antibodies that recognizerelatively conserved regions on the envelope glycoprotein are rare. Thepresent invention is based in part on the stringent characterization ofhomogeneous preparations of trimeric HIV-1 envelope protein in relevantconformations, followed by the analysis of the molecular mechanism ofneutralization by two broadly neutralizing antibodies, 2F5 and 4E10. Itwas discovered that the epitopes of 2F5 and 4E10 are themembrane-proximal segment of the envelope-protein ectodomain, and thatthese epitopes are exposed only on a form of the envelope glycoproteindesigned to mimic an intermediate state during viral entry. Theseresults explain the rarity of 2F5- and 4E10-like antibody responses andindicate a novel strategy for eliciting broadly neutralizing antibodyresponses in a host.

Embodiments of the present invention are directed to scaffolds formaintaining an amino acid sequence or protein, such as membrane-proximalexternal regions, in an immunogenic or antigenic conformation. Accordingto one aspect of the present invention, scaffolds can be altered ordesigned to maintain the same or a substantially similar amino acidsequence or protein in an immunogenic or antigenic conformation.Different scaffold designs can maintain the same amino acid sequence orprotein in an immunogenic or antigenic conformation. In addition, theamino acid sequences or proteins of the present invention can be alteredor modified according to methods known in the art to have differentsequences yet still be capable of being placed in an immunogenic orantigenic conformation. It is to be understood that the specific aminoacid sequences and proteins described herein include sequences andproteins that are substantially similar or homologous thereto or thosethat can be modified in a manner contemplated by those skilled in theart without departing from the spirit and operation of the invention.

Accordingly, the present invention is directed in part to prehairpinintermediate conformations of the envelope protein (e.g., gp41) of ahuman immunodeficiency virus (e.g., HIV-1) and methods for their use. Incertain exemplary embodiments, the compounds and methods describedherein are used to inhibit or decrease one or more HIV-mediatedactivities (e.g., infection, fusion (e.g., target cell entry and/orsyncytia formation), viral spread and the like) in a subject, which can,in turn, decrease HIV titer.

As used herein, the terms “inhibiting” or “decreasing” with respect toHIV refer to an inhibition or decrease of an HIV-mediated activity(e.g., infection, fusion (e.g., target cell entry and/or syncytiaformation), viral spread and the like) and/or a decrease in viral titer.For example, an HIV-mediated activity may be decreased by 5%, 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%,99.8%, 99.9% or more.

HIV is a member of the genus Lentivirinae, part of the family ofRetroviridae. Two species of HIV infect humans: HIV-1 and HIV-2. As usedherein, the terms “human immunodeficiency virus” and “HIV” refer, butare not limited to, HIV-1 and HIV-2. In certain exemplary embodiments,the envelope proteins described herein refer to those present on any ofthe five serogroups of lentiviruses that are recognized: primate (e.g.,HIV-1, HIV-2, simian immunodeficiency virus (SIV)); sheep and goat(e.g., visna virus, caprine arthritis encephalitis virus); horse (equineinfectious anemia virus); cat (e.g., feline immunodeficiency virus(FIV)); and cattle (e.g., bovine immunodeficiency virus (BIV)) (SeeInternational Committee on Taxonomy of Viruses descriptions).

HIV is categorized into multiple clades with a high degree of geneticdivergence. As used herein, the term “clade” refers to related humanimmunodeficiency viruses classified according to their degree of geneticsimilarity. There are currently three groups of HIV-1 isolates: M, N,and O. Group M (major strains) consists of at least ten clades, Athrough J. Group O (outer strains) may consist of a similar number ofclades. Group N is a new HIV-1 isolate that has not been categorized ineither group M or O. In certain exemplary embodiments, a broadlyneutralizing antibody described herein will recognize and raise animmune response against two, three, four, five, six, seven, eight, nine,ten or more clades and/or two or more groups of HIV.

As used herein, the term “envelope glycoprotein” refers, but is notlimited to, the glycoprotein that is expressed on the surface of theenvelope of HIV virions and the surface of the plasma membrane of HIVinfected cells. The env gene encodes gp160, which is proteolyticallycleaved into gp120 and gp140. Gp120 binds to the CD4 receptor on atarget cell that has such a receptor, such as, e.g., a T-helper cell.Gp41 is non-covalently bound to gp120, and provides the second step bywhich HIV enters the cell. It is originally buried within the viralenvelope, but when gp120 binds to a CD4 receptor, gp120 changes itsconformation causing gp41 to become exposed, where it can assist infusion with the host cell.

In certain exemplary embodiments, a prehairpin intermediate conformationof an HIV envelope glycoprotein is provided. As used herein, the term“prehairpin intermediate conformation” refers, but is not limited to,the form of an envelope glycoprotein, e.g., of gp41, that is presentduring the transition from the “prefusion” conformation of the envelopeglycoprotein, as is found on infectious virions, to the “postfusion”conformation, the final, stable conformation after viral entry into atarget cell is complete. In certain aspects, a prehairpin intermediateconformation of an envelope protein includes one, two or more heptadrepeat 1 (HR1) motifs, one, two or more heptad repeat 2 (HR2) motifs,and one, two or more membrane-proximal external regions (MPER). Incertain optional aspects, a prehairpin intermediate conformation of anenvelope protein further includes one or more linker regions. In otheroptional aspects, a prehairpin intermediate conformation of an envelopeprotein includes one or more C-C loop domains. In yet other optionalaspects, a prehairpin intermediate conformation of an envelope proteinincludes one or more oligomerization domains. In certain exemplaryembodiments, a prehairpin intermediate conformation of an envelopeprotein includes, listed in amino terminal to carboxy terminal order:HR2-HR1-HR2-MPER. In certain exemplary embodiments, a prehairpinintermediate conformation of an envelope protein includes, listed inamino terminal to carboxy terminal order: HR2-[optionallinker]-HR1-HR2-MPER; HR2-HR1-HR2-[optional C-C loop domain]-MPER;HR2-HR1-HR2-MPER-[optional oligomerization domain]; orHR2-HR1-HR2-MPER-[optional protein tag]. In certain aspects, aprehairpin intermediate conformation of an envelope protein includesone, two or more oligomerization domains (e.g., trimerization domains),one, two or more HR2 motifs, and one, two or more MPERs. In certainexemplary embodiments, a prehairpin intermediate conformation of anenvelope protein includes, listed in amino terminal to carboxy terminalorder: oligomerization domain-HR2-MPER. In certain exemplaryembodiments, a prehairpin intermediate conformation of an envelopeprotein includes, listed in amino terminal to carboxy terminal order:oligomerization domain-[optional C-C loop domain]-HR2-MPER. In certainexemplary embodiments, a prehairpin intermediate conformation of anenvelope protein includes, listed in amino terminal to carboxy terminalorder: GCN4 trimerization domain-[optional C-C loop domain]-HR2-MPER. Incertain exemplary embodiments, a prehairpin intermediate conformation ofan envelope protein may contain one, two, three or all four of the oneor more optional linkers, optional C-C loop domains, optionaloligomerization domains and optional protein tags. In still otherexemplary embodiments, a prehairpin intermediate conformation of anenvelope protein includes one or more of the specific constructsdescribed further herein (infra).

As used herein, the terms “heptad repeat 1” and “HR1” refer, but are notlimited to, a heptad repeat region that is located at the amino terminusof wild-type gp41. As used herein, the terms “heptad repeat 2” and “HR2”refer, but are not limited to, a heptad repeat region that is located atthe carboxy terminus of wild-type gp41. A heptad repeat is a motif inwhich a hydrophobic amino acid is repeated every seven residues; suchmotifs are designated a through g (Lupas (1996) Trends Biochem. Sci.21:375). Heptad repeats which contain hydrophobic or neutral residues atthe a and d positions can form alpha helices and are able to interactwith other heptad repeats by forming coiled coils (Chambers et al.(1990) J. Gen. Virol. 71:3075; and Lupas, supra). The gp41 HR1 and HR2sequences are well known in the art and are described in, e.g., Milleret al. (2005) Proc. Natl. Acad. Sci. USA 102:14759.

As used herein, the terms “membrane-proximal external region” and “MPER”refer, but are not limited to, a highly conserved region of the gp41ectodomain adjacent to the viral membrane that is well known in the art.

As used herein, the term “C-C loop domain” refers, but is not limitedto, an immunodominant loop present in gp41 proteins that has a conserveddisulfide bond. The HIV C-C loop domain is well known in the art.

As used herein, the terms “linker” and “linker region” refer, but arenot limited to, a polypeptide sequence used to linearly connect twopolypeptide sequences. The linker region may, optionally, addflexibility between the linked polypeptide sequences. A linker regionmay consist of 5, 10, 15, 20, 25, 30 or more amino acid residues. Incertain exemplary embodiments, a linker region comprises serine andglycine residues. In certain exemplary embodiments, a linker regionconsists of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid residues. Incertain exemplary embodiments, a linker region is a short (e.g., lessthan 10 amino acid residues), flexible connector of serines andglycines.

As used herein, the term “oligomerization domain” refers, but is notlimited to, a polypeptide sequence that can be used to increase thestability of an oligomeric envelope protein such as, e.g., to increasethe stability of an HIV gp41 trimer. Oligomerization domains mayincrease the stability of dimers, trimers, tetramers, pentamers,hexamers, heptamers, octamers, nonamers, decamers and larger oligomers.Oligomerization domains can be used to increase the stability ofhomooligomeric polypeptides as well as heterooligomeric polypeptides.Oligomerization domains are well known in the art.

As used herein, the terms “trimerization domain” and “trimerization tag”refer to an oligomerization region that stabilizes trimeric polypeptides(e.g., a gp41 homotrimeric polypeptide). Examples of trimerizationdomain include, but are not limited to, the T4-fibritin “foldon” trimer;the coiled-coil trimer derived from GCN4 (Yang et al. (2002) J. Virol.76:4634); the catalytic subunit of E. coli aspartate transcarbamoylaseas a trimer tag (Chen et al. (2004) J. Virol. 78:4508). Trimerizationdomains are well known in the art.

As used herein, the term “protein tag” refers, but is not limited to, apolypeptide sequence that can be added to another polypeptide sequencefor a variety of purposes. In certain exemplary embodiments, a proteintag may be removed from a larger polypeptide sequence when it is nolonger needed. Protein tags include, but are not limited to, affinitytags (e.g., poly-His tags, chitin binding protein (CBP), maltose bindingprotein (MBP), glutathione-s-transferase (GST) and the like),solubilization tags (e.g., include thioredoxin (TRX), poly(NANP) MBP,GST and the like), chromatography tags (e.g., polyanionic amino acidssuch as the FLAG epitope), epitope tags (e.g., FLAG-tag, V5-tag,c-myc-tag, HA-tag and the like), fluorescent tags (e.g., greenfluorescent protein (GFP), yellow fluorescent protein (YFP), cyanfluorescence protein (CFP) and the like), bioluminescent tags (e.g.,luciferase (e.g., bacterial, firefly, click beetle, sea pansy (Renilla)and the like), luciferin, aequorin and the like), enzyme modificationtags (e.g., biotin ligase and the like) and the like. Protein tags arewell known in the art and their reagents are often commerciallyavailable.

In certain exemplary embodiments, a prehairpin intermediate conformationof an envelope glycoprotein described herein can be administered to asubject in whom it is desirable to promote an immune response. In otherexemplary embodiments, a nucleic acid sequence encoding one or moreprehairpin intermediate conformations of an envelope protein describedherein can be administered to a subject in whom it is desirable topromote an immune response.

Accordingly, one or more prehairpin intermediate conformations ofenvelope glycoprotein(s) can be used as immunogens to produceanti-prehairpin intermediate conformation antibodies in a subject, toinhibit or prevent infection by HIV and/or to inhibit or prevent thespread of HIV in an infected individual. One or more prehairpinintermediate conformations of an envelope glycoprotein described hereincan be used as an immunogen to generate antibodies that bind wild-typeenvelope glycoprotein (i.e., gp41 and/or gp160) using standardtechniques for polyclonal and monoclonal antibody preparation.

In certain exemplary embodiments, a prehairpin intermediate conformationof an envelope glycoprotein is capable of eliciting a broadlyneutralizing antibody response in a host (including, e.g., one or moreof the antibodies described herein (e.g., specific antibodies describedin the Examples)). As used herein, the term “broadly neutralizingantibody response” is well known in the art and refers to the ability ofone or more antibodies to react with an infectious agent to destroy orgreatly reduce the virulence of the infectious agent. The presence ofsuch a response has the potential to prevent the establishment ofinfection and/or to significantly reduce the number of cells that becomeinfected with HIV, potentially delaying viral spread and allowing for abetter control of viral replication in the infected host. A broadlyneutralizing antibody against HIV will typically bind a variety ofdifferent clades, groups or mutants of HIV.

As used herein, the term “immune response” is intended to include, butis not limited to, T and/or B cell responses, that is, cellular and/orhumoral immune responses. The immune response of a subject can bedetermined by, for example, assaying antibody production, immune cellproliferation, the release of cytokines, the expression of cell surfacemarkers, cytotoxicity, and the like. As used herein, the term “immunecell” is intended to include, but is not limited to, cells that are ofhematopoietic origin and play a role in an immune response. Immune cellsinclude, but are not limited to, lymphocytes, such as B cells and Tcells; natural killer cells; myeloid cells, such as monocytes,macrophages, eosinophils, mast cells, basophils, and granulocytes.

A prehairpin intermediate conformation of an envelope glycoproteintypically is used to prepare antibodies by immunizing a suitablesubject, (e.g., rabbit, goat, mouse or other mammal) with the immunogen.An appropriate immunogenic preparation can contain, for example, arecombinantly expressed prehairpin intermediate conformation of anenvelope glycoprotein or a chemically synthesized prehairpinintermediate conformation of an envelope glycoprotein. The preparationcan further include an adjuvant, such as Freund's complete or incompleteadjuvant, or similar immunostimulatory agent. Immunization of a suitablesubject with an immunogenic prehairpin intermediate conformation of anenvelope glycoprotein preparation induces a polyclonal anti-envelope(e.g., anti-gp41 and/or anti-gp160) antibody response, e.g., an anti-HIVantibody response.

Accordingly, in certain exemplary embodiments, anti-prehairpinintermediate conformation of gp41 antibodies are provided. The term“antibody” as used herein refers to immunoglobulin molecules andimmunologically active portions of immunoglobulin molecules, i.e.,molecules that contain an antigen binding site which specifically binds(immunoreacts with) an antigen, such as the envelope glycoprotein (e.g.,gp41 and/or gp160). Examples of immunologically active portions ofimmunoglobulin molecules include F(ab) and F(ab′)2 fragments which canbe generated by treating the antibody with an enzyme such as pepsin. Theinvention provides polyclonal and monoclonal antibodies that bind theenvelope glycoprotein (e.g., gp41 and/or gp160). The term “monoclonalantibody” or “monoclonal antibody composition,” as used herein, refersto a population of antibody molecules that contain only one species ofan antigen binding site capable of immunoreacting with a particularepitope of the envelope glycoprotein (e.g., gp41 and/or gp160). Amonoclonal antibody composition thus typically displays a single bindingaffinity for a particular the envelope glycoprotein (e.g., gp41 and/orgp160) with which it immunoreacts.

Polyclonal anti-envelope glycoprotein (e.g., gp41 and/or gp160)antibodies can be prepared as described above by immunizing a suitablesubject with a prehairpin intermediate conformation of an envelopeglycoprotein immunogen as described herein. The anti-prehairpinintermediate conformation of an envelope glycoprotein antibody titer inthe immunized subject can be monitored over time by standard techniques,such as with an enzyme linked immunosorbent assay (ELISA) usingimmobilized gp41. If desired, the antibody molecules directed againstgp41 can be isolated from the mammal (e.g., from the blood) and furtherpurified by well known techniques, such as protein A chromatography toobtain the IgG fraction. At an appropriate time after immunization,e.g., when the anti-gp41 antibody titers are highest, antibody-producingcells can be obtained from the subject and used to prepare monoclonalantibodies by standard techniques, such as the hybridoma techniqueoriginally described by Kohler and Milstein (1975) Nature 256:495-497)(see also, Brown et al. (1981) J. Immunol. 127:539-46; Brown et al.(1980) J. Biol. Chem. 255:4980-83; Yeh et al. (1976) Proc. Natl. Acad.Sci. USA 76:2927-31; and Yeh et al. (1982) Int. J. Cancer 29:269-75),the human B cell hybridoma technique (Kozbor et al. (1983) Immunol.Today 4:72), the EBV-hybridoma technique (Cole et al. (1985), MonoclonalAntibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96) or triomatechniques. The technology for producing monoclonal antibody hybridomasis well known (see generally R. H. Kenneth, in Monoclonal Antibodies: ANew Dimension In Biological Analyses, Plenum Publishing Corp., New York,N.Y. (1980); E. A. Lerner (1981) Yale J. Biol. Med. 54:387-402; Gefteret al. (1977) Somatic Cell Genet. 3:231-36). Briefly, an immortal cellline (typically a myeloma) is fused to lymphocytes (typicallysplenocytes) from a mammal immunized with a prehairpin intermediateconformation of an envelope glycoprotein immunogen as described above,and the culture supernatants of the resulting hybridoma cells arescreened to identify a hybridoma producing a monoclonal antibody thatbinds gp41.

Any of the many well known protocols used for fusing lymphocytes andimmortalized cell lines can be applied for the purpose of generating ananti-prehairpin intermediate conformation of an envelope glycoproteinmonoclonal antibody (see, e.g., G. Galfre et al. (1977) Nature266:55052; Gefter et al. Somatic Cell Genet., cited supra; Lerner, YaleJ. Biol. Med. (supra); Kenneth, Monoclonal Antibodies, (supra)).Moreover, the ordinarily skilled worker will appreciate that there aremany variations of such methods which also would be useful. Typically,the immortal cell line (e.g., a myeloma cell line) is derived from thesame mammalian species as the lymphocytes. For example, murinehybridomas can be made by fusing lymphocytes from a mouse immunized withan immunogenic preparation of the present invention with an immortalizedmouse cell line. Particularly suitable immortal cell lines are mousemyeloma cell lines that are sensitive to culture medium containinghypoxanthine, aminopterin and thymidine (“HAT medium”). Any of a numberof myeloma cell lines can be used as a fusion partner according tostandard techniques, e.g., the P3-NS1/1-Ag4-1, P3-x63-Ag8.653 orSp2/O—Ag14 myeloma lines. These myeloma lines are available from ATCC.Typically, HAT-sensitive mouse myeloma cells are fused to mousesplenocytes using polyethylene glycol (“PEG”). Hybridoma cells resultingfrom the fusion are then selected using HAT medium, which kills unfusedand unproductively fused myeloma cells (unfused splenocytes die afterseveral days because they are not transformed). Hybridoma cellsproducing a monoclonal antibody of a prehairpin intermediateconformation of an envelope glycoprotein are detected by screening thehybridoma culture supernatants for antibodies that bind gp41, e.g.,using a standard ELISA assay.

Alternative to preparing monoclonal antibody-secreting hybridomas, amonoclonal anti-prehairpin intermediate conformation of an envelopeglycoprotein antibody can be identified and isolated by screening arecombinant combinatorial immunoglobulin library (e.g., an antibodyphage display library) with a gp41 protein to thereby isolateimmunoglobulin library members that bind gp41. Kits for generating andscreening phage display libraries are commercially available (e.g.,Recombinant Phage Antibody System, Pfizer, New York, N.Y.; and theSURFZAP™ Phage Display Kit, Stratagene, La Jolla, Calif.). Additionally,examples of methods and reagents particularly amenable for use ingenerating and screening antibody display library can be found in, forexample, Ladner et al. U.S. Pat. No. 5,223,409; Kang et al. PCTInternational Publication No. WO 92/18619; Dower et al. PCTInternational Publication No. WO 91/17271; Winter et al. PCTInternational Publication WO 92/20791; Markland et al. PCT InternationalPublication No. WO 92/15679; Breitling et al. PCT InternationalPublication WO93/01288; McCafferty et al. PCT International PublicationNo. WO 92/01047; Garrard et al. PCT International Publication No. WO92/09690; Ladner et al. PCT International Publication No. WO 90/02809;Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay et al. (1992) Hum.Antibod. Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281;Griffiths et al. (1993) EMBO J. 12:725-734; Hawkins et al. (1992) J.Mol. Biol. 226:889-896; Clarkson et al. (1991) Nature 352:624-628; Gramet al. (1992) Proc. Natl. Acad. Sci. USA 89:3576-3580; Garrad et al.(1991) Bio/Technology 9:1373-1377; Hoogenboom et al. (1991) Nucl. AcidRes. 19:4133-4137; Barbas et al. (1991) Proc. Natl. Acad. Sci. USA88:7978-7982; and McCafferty et al. (1990) Nature 348:552-554.

Additionally, recombinant anti-prehairpin intermediate conformations ofenvelope glycoprotein antibodies, such as chimeric and humanizedmonoclonal antibodies, comprising both human and non-human portions,which can be made using standard recombinant DNA techniques, are withinthe scope of the invention. Such chimeric and humanized monoclonalantibodies can be produced by recombinant DNA techniques known in theart, for example using methods described in Robinson et al.International Application No. PCT/US86/02269; Akira, et al. EuropeanPatent Application 184,187; Taniguchi, M., European Patent Application171,496; Morrison et al. European Patent Application 173,494; Neubergeret al. PCT International Publication No. WO 86/01533; Cabilly et al.U.S. Pat. No. 4,816,567; Cabilly et al. European Patent Application125,023; Better et al. (1988) Science 240:1041-1043; Liu et al. (1987)Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al. (1987) J. Immunol.139:3521-3526; Sun et al. (1987) Proc. Natl. Acad. Sci. USA 84:214-218;Nishimura et al. (1987) Canc. Res. 47:999-1005; Wood et al. (1985)Nature 314:446-449; and Shaw et al. (1988) J. Natl. Cancer Inst.80:1553-1559); Morrison, S. L. (1985) Science 229:1202-1207; Oi et al.(1986) BioTechniques 4:214; Winter U.S. Pat. No. 5,225,539; Jones et al.(1986) Nature 321:552-525; Verhoeyan et al. (1988) Science 239:1534; andBeidler et al. (1988) J. Immunol. 141:4053-4060.

In certain exemplary embodiments, antibodies, fusion inhibiting agents(e.g., small molecules, peptides and the like) and the like that arecapable of interacting with a prehairpin intermediate conformation of anHIV envelope glycoprotein are provided. As used herein, the terms“bind,” “binding,” “interact,” “interacting,” “occupy” and “occupying”refer to covalent interactions, noncovalent interactions and stericinteractions. A covalent interaction is a chemical linkage between twoatoms or radicals formed by the sharing of a pair of electrons (a singlebond), two pairs of electrons (a double bond) or three pairs ofelectrons (a triple bond). Covalent interactions are also known in theart as electron pair interactions or electron pair bonds. Noncovalentinteractions include, but are not limited to, van der Waalsinteractions, hydrogen bonds, weak chemical bonds (via short-rangenoncovalent forces), hydrophobic interactions, ionic bonds and the like.A review of noncovalent interactions can be found in Alberts et al., inMolecular Biology of the Cell, 3d edition, Garland Publishing, 1994.Steric interactions are generally understood to include those where thestructure of the compound is such that it is capable of occupying a siteby virtue of its three dimensional structure, as opposed to anyattractive forces between the compound and the site.

In certain exemplary embodiments, compositions and methods for enhancingthe immune response of a subject to a human immunodeficiency virus areprovided. As used herein, the terms “subject” and “host” are intended toinclude living organisms such as mammals. Examples of subjects and hostsinclude, but are not limited to, horses, cows, sheep, pigs, goats, dogs,cats, rabbits, guinea pigs, rats, mice, gerbils, non-human primates(e.g., macaques), humans and the like, non-mammals, including, e.g.,non-mammalian vertebrates, such as birds (e.g., chickens or ducks) fishor frogs (e.g., Xenopus), and non-mammalian invertebrates, as well astransgenic species thereof.

In certain exemplary embodiments, vectors such as, for example,expression vectors, containing a nucleic acid encoding one or moreprehairpin intermediate conformations of an envelope protein describedherein are provided. As used herein, the term “vector” refers to anucleic acid molecule capable of transporting another nucleic acid towhich it has been linked. One type of vector is a “plasmid,” whichrefers to a circular double stranded DNA loop into which additional DNAsegments can be ligated. Another type of vector is a viral vector,wherein additional DNA segments can be ligated into the viral genome.Certain vectors are capable of autonomous replication in a host cellinto which they are introduced (e.g., bacterial vectors having abacterial origin of replication and episomal mammalian vectors). Othervectors (e.g., non-episomal mammalian vectors) are integrated into thegenome of a host cell upon introduction into the host cell, and therebyare replicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to herein as “expressionvectors.” In general, expression vectors of utility in recombinant DNAtechniques are often in the form of plasmids. In the presentspecification, “plasmid” and “vector” can be used interchangeably.However, the invention is intended to include such other forms ofexpression vectors, such as viral vectors (e.g., replication defectiveretroviruses, adenoviruses and adeno-associated viruses), which serveequivalent functions.

In certain exemplary embodiments, the recombinant expression vectorscomprise a nucleic acid sequence (e.g., a nucleic acid sequence encodingone or more prehairpin intermediate conformations of an envelope proteindescribed herein) in a form suitable for expression of the nucleic acidsequence in a host cell, which means that the recombinant expressionvectors include one or more regulatory sequences, selected on the basisof the host cells to be used for expression, which is operatively linkedto the nucleic acid sequence to be expressed. Within a recombinantexpression vector, “operably linked” is intended to mean that thenucleotide sequence encoding one or more prehairpin intermediateconformations of an envelope protein is linked to the regulatorysequence(s) in a manner which allows for expression of the nucleotidesequence (e.g., in an in vitro transcription/translation system or in ahost cell when the vector is introduced into the host cell). The term“regulatory sequence” is intended to include promoters, enhancers andother expression control elements (e.g., polyadenylation signals). Suchregulatory sequences are described, for example, in Goeddel; GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990). Regulatory sequences include those which directconstitutive expression of a nucleotide sequence in many types of hostcells and those which direct expression of the nucleotide sequence onlyin certain host cells (e.g., tissue-specific regulatory sequences). Itwill be appreciated by those skilled in the art that the design of theexpression vector can depend on such factors as the choice of the hostcell to be transformed, the level of expression of protein desired, andthe like. The expression vectors described herein can be introduced intohost cells to thereby produce proteins or portions thereof, includingfusion proteins or portions thereof, encoded by nucleic acids asdescribed herein (e.g., one or more prehairpin intermediateconformations of an envelope protein).

In certain exemplary embodiments, nucleic acid molecules describedherein can be inserted into vectors and used as gene therapy vectors.Gene therapy vectors can be delivered to a subject by, for example,intravenous injection, local administration (see, e.g., U.S. Pat. No.5,328,470), or by stereotactic injection (see, e.g., Chen et al. (1994)Proc. Natl. Acad. Sci. U.S.A. 91:3054). The pharmaceutical preparationof the gene therapy vector can include the gene therapy vector in anacceptable diluent, or can comprise a slow release matrix in which thegene delivery vehicle is imbedded. Alternatively, where the completegene delivery vector can be produced intact from recombinant cells,e.g., retroviral vectors, adeno-associated virus vectors, and the like,the pharmaceutical preparation can include one or more cells whichproduce the gene delivery system (See Gardlik et al. (2005) Med. Sci.Mon. 11:110; Salmons and Gunsberg (1993) Hu. Gene Ther. 4:129; and Wanget al. (2005) J. Virol. 79:10999 for reviews of gene therapy vectors).

Recombinant expression vectors of the invention can be designed forexpression of one or more encoding one or more prehairpin intermediateconformations of an envelope protein in prokaryotic or eukaryotic cells.For example, one or more vectors encoding one or more prehairpinintermediate conformations of an envelope protein can be expressed inbacterial cells such as E. coli, insect cells (e.g., using baculovirusexpression vectors), yeast cells or mammalian cells. Suitable host cellsare discussed further in Goeddel, Gene Expression Technology: Methods inEnzymology 185, Academic Press, San Diego, Calif. (1990). Alternatively,the recombinant expression vector can be transcribed and translated invitro, for example using T7 promoter regulatory sequences and T7polymerase.

Expression of proteins in prokaryotes is most often carried out in E.coli with vectors containing constitutive or inducible promotersdirecting the expression of either fusion or non-fusion proteins. Fusionvectors add a number of amino acids to a protein encoded therein,usually to the amino terminus of the recombinant protein. Such fusionvectors typically serve three purposes: 1) to increase expression ofrecombinant protein; 2) to increase the solubility of the recombinantprotein; and 3) to aid in the purification of the recombinant protein byacting as a ligand in affinity purification. Often, in fusion expressionvectors, a proteolytic cleavage site is introduced at the junction ofthe fusion moiety and the recombinant protein to enable separation ofthe recombinant protein from the fusion moiety subsequent topurification of the fusion protein. Such enzymes, and their cognaterecognition sequences, include Factor Xa, thrombin and enterokinase.Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc;Smith, D. B. and Johnson, K. S. (1988) Gene 67:31-40); pMAL (New EnglandBiolabs, Beverly, Mass.); and pRIT5 (Pharmacia, Piscataway, N.J.) whichfuse glutathione S-transferase (GST), maltose E binding protein, orprotein A, respectively, to the target recombinant protein.

In another embodiment, the expression vector encoding one or moreprehairpin intermediate conformations of an envelope protein is a yeastexpression vector. Examples of vectors for expression in yeast S.cerevisiae include pYepSec1 (Baldari, et. al., (1987) EMBO J.6:229-234); pMFa (Kurjan and Herskowitz, (1982) Cell 30:933-943); pJRY88(Schultz et al., (1987) Gene 54:113-123); pYES2 (Invitrogen Corporation,San Diego, Calif.); and picZ (Invitrogen Corporation).

Alternatively, one or more prehairpin intermediate conformations of anenvelope protein can be expressed in insect cells using baculovirusexpression vectors. Baculovirus vectors available for expression ofproteins in cultured insect cells (e.g., Sf9 cells) include the pAcseries (Smith et al. (1983) Mol. Cell. Biol. 3:2156-2165) and the pVLseries (Lucklow and Summers (1989) Virology 170:31-39).

In certain exemplary embodiments, a nucleic acid described herein isexpressed in mammalian cells using a mammalian expression vector.Examples of mammalian expression vectors include pCDM8 (Seed, B. (1987)Nature 329:840) and pMT2PC (Kaufman et al. (1987) EMBO J. 6:187-195).When used in mammalian cells, the expression vector's control functionsare often provided by viral regulatory elements. For example, commonlyused promoters are derived from polyoma, adenovirus 2, cytomegalovirusand simian virus 40. For other suitable expression systems for bothprokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook, J.,Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual.2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989.

In certain exemplary embodiments, the recombinant mammalian expressionvector is capable of directing expression of the nucleic acidpreferentially in a particular cell type (e.g., tissue-specificregulatory elements are used to express the nucleic acid).Tissue-specific regulatory elements are known in the art. Non-limitingexamples of suitable tissue-specific promoters include lymphoid-specificpromoters (Calame and Eaton (1988) Adv. Immunol. 43:235), in particularpromoters of T cell receptors (Winoto and Baltimore (1989) EMBO J.8:729) and immunoglobulins (Banerji et al. (1983) Cell 33:729; Queen andBaltimore (1983) Cell 33:741), neuron-specific promoters (e.g., theneurofilament promoter; Byrne and Ruddle (1989) Proc. Natl. Acad. Sci.U.S.A. 86:5473), pancreas-specific promoters (Edlund et al. (1985)Science 230:912), and mammary gland-specific promoters (e.g., milk wheypromoter; U.S. Pat. No. 4,873,316 and European Application PublicationNo. 264,166). Developmentally-regulated promoters are also encompassed,for example the murine hox promoters (Kessel and Gruss (1990) Science249:374) and the α-fetoprotein promoter (Campes and Tilghman (1989)Genes Dev. 3:537).

In certain exemplary embodiments, host cells into which a recombinantexpression vector of the invention has been introduced are provided. Theterms “host cell” and “recombinant host cell” are used interchangeablyherein. It is understood that such terms refer not only to theparticular subject cell but to the progeny or potential progeny of sucha cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein.

A host cell can be any prokaryotic or eukaryotic cell. For example, oneor more prehairpin intermediate conformations of an envelope protein canbe expressed in bacterial cells such as E. coli, viral cells such asretroviral cells, insect cells, yeast or mammalian cells (such asChinese hamster ovary cells (CHO) or COS cells). Other suitable hostcells are known to those skilled in the art.

Delivery of nucleic acids described herein (e.g., vector DNA) can be byany suitable method in the art. For example, delivery may be byinjection, gene gun, by application of the nucleic acid in a gel, oil,or cream, by electroporation, using lipid-based transfection reagents,or by any other suitable transfection method.

As used herein, the terms “transformation” and “transfection” areintended to refer to a variety of art-recognized techniques forintroducing foreign nucleic acid (e.g., DNA) into a host cell, includingcalcium phosphate or calcium chloride co-precipitation,DEAE-dextran-mediated transfection, lipofection (e.g., usingcommercially available reagents such as, for example, LIPOFECTIN®(Invitrogen Corp., San Diego, Calif.), LIPOFECTAMINE® (Invitrogen),FUGENE® (Roche Applied Science, Basel, Switzerland), JETPEI®(Polyplus-transfection Inc., New York, N.Y.), EFFECTENE® (Qiagen,Valencia, Calif.), DREAMFECT® (OZ Biosciences, France) and the like), orelectroporation (e.g., in vivo electroporation). Suitable methods fortransforming or transfecting host cells can be found in Sambrook, et al.(Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring harborLaboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 1989), and other laboratory manuals.

Embodiments of the invention are directed to a first nucleic acid (e.g.,a nucleic acid sequence encoding one or more gp41 domains or motifs suchas, for example, HR1 from a wild type gp41 strain, HR2 from a wild typegp41 strain, MPER from a wild type gp41 strain and the like) orpolypeptide sequence (e.g., one or more gp41 domains or motifs such as,for example, HR1 from a wild type gp41 strain, HR2 from a wild type gp41strain, MPER from a wild type gp41 strain and the like) having a certainsequence identity or percent homology to a second nucleic acid orpolypeptide sequence, respectively.

Techniques for determining nucleic acid and amino acid “sequenceidentity” are known in the art. Typically, such techniques includedetermining the nucleotide sequence of genomic DNA, mRNA or cDNA madefrom an mRNA for a gene and/or determining the amino acid sequence thatit encodes, and comparing one or both of these sequences to a secondnucleotide or amino acid sequence, as appropriate. In general,“identity” refers to an exact nucleotide-to-nucleotide or aminoacid-to-amino acid correspondence of two polynucleotides or polypeptidesequences, respectively. Two or more sequences (polynucleotide or aminoacid) can be compared by determining their “percent identity.” Thepercent identity of two sequences, whether nucleic acid or amino acidsequences, is the number of exact matches between two aligned sequencesdivided by the length of the shorter sequences and multiplied by 100.

An approximate alignment for nucleic acid sequences is provided by thelocal homology algorithm of Smith and Waterman, Advances in AppliedMathematics 2:482-489 (1981). This algorithm can be applied to aminoacid sequences by using the scoring matrix developed by Dayhoff, Atlasof Protein Sequences and Structure, M. O. Dayhoff ed., 5 suppl.3:353-358, National Biomedical Research Foundation, Washington, D.C.,USA, and normalized by Gribskov (1986) Nucl. Acids Res. 14:6745. Anexemplary implementation of this algorithm to determine percent identityof a sequence is provided by the Genetics Computer Group (Madison, Wis.)in the “BestFit” utility application. The default parameters for thismethod are described in the Wisconsin Sequence Analysis Package ProgramManual, Version 8 (1995) (available from Genetics Computer Group,Madison, Wis.).

One method of establishing percent identity in the context of thepresent invention is to use the MPSRCH package of programs copyrightedby the University of Edinburgh, developed by John F. Collins and ShaneS. Sturrok, and distributed by IntelliGenetics, Inc. (Mountain View,Calif.). From this suite of packages, the Smith-Waterman algorithm canbe employed where default parameters are used for the scoring table (forexample, gap open penalty of 12, gap extension penalty of one, and a gapof six). From the data generated the “match” value reflects “sequenceidentity.” Other suitable programs for calculating the percent identityor similarity between sequences are generally known in the art, forexample, another alignment program is BLAST, used with defaultparameters. For example, BLASTN and BLASTP can be used using thefollowing default parameters: genetic code=standard; filter=none;strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50sequences; sort by ═HIGH SCORE; Databases=non-redundant,GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+Swissprotein+Spupdate+PIR. Details of these programs can be found at theNCBI/NLM web site.

Alternatively, homology can be determined by hybridization ofpolynucleotides under conditions that form stable duplexes betweenhomologous regions, followed by digestion with single-stranded-specificnuclease(s), and size determination of the digested fragments. Two DNAsequences, or two polypeptide sequences are “substantially homologous”to each other when the sequences exhibit at least about 80%-85%, atleast about 85%-90%, at least about 90%-95%, or at least about 95%-98%sequence identity over a defined length of the molecules, as determinedusing the methods above. As used herein, substantially homologous alsorefers to sequences showing complete identity to the specified DNA orpolypeptide sequence. DNA sequences that are substantially homologouscan be identified in a Southern hybridization experiment under, forexample, stringent conditions, as defined for that particular system.Defining appropriate hybridization conditions is within the skill of theart. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual,Second Edition, (1989) Cold Spring Harbor, N.Y.; Nucleic AcidHybridization: A Practical Approach, editors B. D. Hames and S. J.Higgins, (1985) Oxford; Washington, D.C.; IRL Press.

Two nucleic acid fragments are considered to “selectively hybridize” asdescribed herein. The degree of sequence identity between two nucleicacid molecules affects the efficiency and strength of hybridizationevents between such molecules. A partially identical nucleic acidsequence will at least partially inhibit a completely identical sequencefrom hybridizing to a target molecule. Inhibition of hybridization ofthe completely identical sequence can be assessed using hybridizationassays that are well known in the art (e.g., Southern blot, Northernblot, solution hybridization, or the like, see Sambrook, et al., supra).Such assays can be conducted using varying degrees of selectivity, forexample, using conditions varying from low to high stringency. Ifconditions of low stringency are employed, the absence of non-specificbinding can be assessed using a secondary probe that lacks even apartial degree of sequence identity (for example, a probe having lessthan about 30% sequence identity with the target molecule), such that,in the absence of non-specific binding events, the secondary probe willnot hybridize to the target.

When utilizing a hybridization-based detection system, a nucleic acidprobe is chosen that is complementary to a target nucleic acid sequence,and then by selection of appropriate conditions the probe and the targetsequence “selectively hybridize,” or bind, to each other to form ahybrid molecule. A nucleic acid molecule that is capable of hybridizingselectively to a target sequence under “moderately stringent” conditionstypically hybridizes under conditions that allow detection of a targetnucleic acid sequence of at least about 10-14 nucleotides in lengthhaving at least approximately 70% sequence identity with the sequence ofthe selected nucleic acid probe. Stringent hybridization conditionstypically allow detection of target nucleic acid sequences of at leastabout 10-14 nucleotides in length having a sequence identity of greaterthan about 90-95% with the sequence of the selected nucleic acid probe.Hybridization conditions useful for probe/target hybridization where theprobe and target have a specific degree of sequence identity, can bedetermined as is known in the art (see, for example, Nucleic AcidHybridization, supra).

With respect to stringency conditions for hybridization, it is wellknown in the art that numerous equivalent conditions can be employed toestablish a particular stringency by varying, for example, the followingfactors: the length and nature of probe and target sequences, basecomposition of the various sequences, concentrations of salts and otherhybridization solution components, the presence or absence of blockingagents in the hybridization solutions (e.g., formamide, dextran sulfate,and polyethylene glycol), hybridization reaction temperature and timeparameters, as well as varying wash conditions. The selection of aparticular set of hybridization conditions is selected followingstandard methods in the art (see, for example, Sambrook et al., supra).

As used herein, the term “hybridizes under stringent conditions” isintended to describe conditions for hybridization and washing underwhich nucleotide sequences at least 60% identical to each othertypically remain hybridized to each other. In one aspect, the conditionsare such that sequences at least about 70%, at least about 80%, at leastabout 85% or 90% or more identical to each other typically remainhybridized to each other. Such stringent conditions are known to thoseskilled in the art and can be found in Current Protocols in MolecularBiology, John Wiley & Sons, NY (1989), 6.3.1-6.3.6. A non-limitingexample of stringent hybridization conditions are hybridization in 6×sodium chloride/sodium citrate (SSC) at about 45° C., followed by one ormore washes in 0.2×SSC, 0.1% SDS at 50° C., at 55° C., or at 60° C. or65° C.

A first polynucleotide is “derived from” a second polynucleotide if ithas the same or substantially the same base-pair sequence as a region ofthe second polynucleotide, its cDNA, complements thereof, or if itdisplays sequence identity as described above. A first polypeptide isderived from a second polypeptide if it is encoded by a firstpolynucleotide derived from a second polynucleotide, or displayssequence identity to the second polypeptides as described above. In thepresent invention, when a gp41 protein is “derived from HIV” the gp41protein need not be explicitly produced by the virus itself, the virusis simply considered to be the original source of the gp41 proteinand/or nucleic acid sequences that encode it. Gp41 proteins can, forexample, be produced recombinantly or synthetically, by methods known inthe art, or alternatively, gp41 proteins may be purified fromHIV-infected cell cultures.

In certain exemplary embodiments screening assays for identifyingmodulators, i.e., candidate or test compounds or agents (e.g.,antibodies, peptides, cyclic peptides, peptidomimetics, small molecules,small organic molecules, or other drugs) which have an inhibitory effecton gp41 and/or one or more HIV mediated activities described herein(e.g., one or more prehairpin intermediate conformations of an envelopeprotein) are provided.

As used herein, the term “small molecule” refers to a molecule, eithernaturally occurring or synthetic, that has a molecular weight of morethan about 25 daltons and less than about 3000 daltons, usually lessthan about 2500 daltons, more usually less than about 2000 daltons,usually between about 100 to about 1000 daltons, more usually betweenabout 200 to about 500 daltons.

In certain exemplary embodiments, assays for screening candidate or testcompounds which bind to or modulate (e.g., inhibit) one or moreprehairpin intermediate conformations of an envelope protein areprovided. The test compounds of the present invention can be obtainedusing any of the numerous approaches in combinatorial library methodsknown in the art, including: biological libraries; spatially addressableparallel solid phase or solution phase libraries; synthetic librarymethods requiring deconvolution; the “one-bead one-compound” librarymethod; and synthetic library methods using affinity chromatographyselection. The biological library approach is limited to peptidelibraries, while the other four approaches are applicable to peptide,non-peptide oligomer or small molecule libraries of compounds (Lam, K.S. (1997) Anticancer Drug Des. 12:145).

The test compound(s), antibodies, one or more prehairpin intermediateconformations of an envelope protein and/or nucleic acid sequencesencoding one or more prehairpin intermediate conformations of anenvelope protein described herein can be incorporated intopharmaceutical compositions suitable for administration. Suchcompositions typically comprise the nucleic acid molecule or protein anda pharmaceutically acceptable carrier. As used herein the language“pharmaceutically acceptable carrier” is intended to include any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like,compatible with pharmaceutical administration. The use of such media andagents for pharmaceutically active substances is well known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the compositions is contemplated.Supplementary active compounds can also be incorporated into thecompositions.

In certain exemplary embodiments, a pharmaceutical composition isformulated to be compatible with its intended route of administration.Examples of routes of administration include parenteral, e.g.,intravenous, intradermal, subcutaneous, oral (e.g., inhalation),transdermal (topical), transmucosal, and rectal administration.Solutions or suspensions used for parenteral, intradermal, orsubcutaneous application can include the following components: a sterilediluent such as water for injection, saline solution, fixed oils,polyethylene glycols, glycerin, propylene glycol or other syntheticsolvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas acetates, citrates or phosphates and agents for the adjustment oftonicity such as sodium chloride or dextrose. pH can be adjusted withacids or bases, such as hydrochloric acid or sodium hydroxide. Theparenteral preparation can be enclosed in ampoules, disposable syringesor multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CREMOPHOREL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the testcompound(s), one or more antibodies, one or more prehairpin intermediateconformations of an envelope protein and/or nucleic acid sequencesencoding one or more prehairpin intermediate conformations of anenvelope protein described herein in the required amount in anappropriate solvent with one or a combination of ingredients enumeratedabove, as required, followed by filtered sterilization. Generally,dispersions are prepared by incorporating the active compound into asterile vehicle which contains a basic dispersion medium and therequired other ingredients from those enumerated above. In the case ofsterile powders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and freeze-dryingwhich yields a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: A binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic, acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant: such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

In one embodiment, the test compound(s), one or more antibodies, one ormore prehairpin intermediate conformations of an envelope protein and/ornucleic acid sequences encoding one or more prehairpin intermediateconformations of an envelope protein described herein are prepared withcarriers that will protect the compound against rapid elimination fromthe body, such as a controlled release formulation, including implantsand microencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These may be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

Nasal compositions generally include nasal sprays and inhalants. Nasalsprays and inhalants can contain one or more active components andexcipients such as preservatives, viscosity modifiers, emulsifiers,buffering agents and the like. Nasal sprays may be applied to the nasalcavity for local and/or systemic use. Nasal sprays may be dispensed by anon-pressurized dispenser suitable for delivery of a metered dose of theactive component. Nasal inhalants are intended for delivery to the lungsby oral inhalation for local and/or systemic use. Nasal inhalants may bedispensed by a closed container system for delivery of a metered dose ofone or more active components.

In one embodiment, nasal inhalants are used with an aerosol. This isaccomplished by preparing an aqueous aerosol, liposomal preparation orsolid particles containing the compound. A non-aqueous (e.g.,fluorocarbon propellant) suspension could be used. Sonic nebulizers maybe used to minimize exposing the agent to shear, which can result indegradation of the compound.

Ordinarily, an aqueous aerosol is made by formulating an aqueoussolution or suspension of the agent together with conventionalpharmaceutically acceptable carriers and stabilizers. The carriers andstabilizers vary with the requirements of the particular compound, buttypically include nonionic surfactants (Tweens, Pluronics, orpolyethylene glycol), innocuous proteins like serum albumin, sorbitanesters, oleic acid, lecithin, amino acids such as glycine, buffers,salts, sugars or sugar alcohols. Aerosols generally are prepared fromisotonic solutions.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The test compound(s), one or more antibodies, one or more prehairpinintermediate conformations of an envelope protein and/or nucleic acidsequences encoding one or more prehairpin intermediate conformations ofan envelope protein described herein can also be prepared in the form ofsuppositories (e.g., with conventional suppository bases such as cocoabutter and other glycerides) or retention enemas for rectal delivery.

In one embodiment, the test compound(s), one or more antibodies, one ormore prehairpin intermediate conformations of an envelope protein and/ornucleic acid sequences encoding one or more one or more prehairpinintermediate conformations of an envelope protein described herein areprepared with carriers that will protect them against rapid eliminationfrom the body, such as a controlled release formulation, includingimplants and microencapsulated delivery systems. Biodegradable,biocompatible polymers can be used, such as ethylene vinyl acetate,polyanhydrides, polyglycolic acid, collagen, polyorthoesters, andpolylactic acid. Methods for preparation of such formulations will beapparent to those skilled in the art. The materials can also be obtainedcommercially from Alza Corporation and Nova Pharmaceuticals, Inc.Liposomal suspensions (including liposomes targeted to infected cellswith monoclonal antibodies to viral antigens) can also be used aspharmaceutically acceptable carriers. These can be prepared according tomethods known to those skilled in the art, for example, as described inU.S. Pat. No. 4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

Toxicity and therapeutic efficacy of the test compound(s), one or moreantibodies, one or more prehairpin intermediate conformations of anenvelope protein and/or nucleic acid sequences encoding one or more oneor more prehairpin intermediate conformations of an envelope proteindescribed herein can be determined by standard pharmaceutical proceduresin cell cultures or experimental animals, e.g., for determining the LD50(the dose lethal to 50% of the population) and the ED50 (the dosetherapeutically effective in 50% of the population). The dose ratiobetween toxic and therapeutic effects is the therapeutic index and itcan be expressed as the ratio LD50/ED50. Compounds which exhibit largetherapeutic indices are preferred. While compounds that exhibit toxicside effects may be used, care should be taken to design a deliverysystem that targets such compounds to the site of affected tissue inorder to minimize potential damage to uninfected cells and, thereby,reduce side effects.

Data obtained from cell culture assays and/or animal studies can be usedin formulating a range of dosage for use in humans. The dosage typicallywill lie within a range of circulating concentrations that include theED50 with little or no toxicity. The dosage may vary within this rangedepending upon the dosage form employed and the route of administrationutilized. For any compound used in the method of the invention, thetherapeutically effective dose can be estimated initially from cellculture assays. A dose may be formulated in animal models to achieve acirculating plasma concentration range that includes the IC50 (i.e., theconcentration of the test compound which achieves a half-maximalinhibition of symptoms) as determined in cell culture. Such informationcan be used to more accurately determine useful doses in humans. Levelsin plasma may be measured, for example, by high performance liquidchromatography.

In certain exemplary embodiments, a method for treatment of a viralinfection, e.g., HIV infection, includes the step of administering atherapeutically effective amount of an agent (e.g., one or more testcompounds, one or more antibodies, one or more prehairpin intermediateconformations of an envelope protein, a nucleic acid sequence thatencodes one or more prehairpin intermediate conformations of an envelopeprotein and the like) which modulates (e.g., inhibits), one or moreenvelope protein (e.g., gp41) activities (e.g., mediating viral fusion(e.g., viral entry and/or syncytia formation)) to a subject. As definedherein, a therapeutically effective amount of agent (i.e., an effectivedosage) ranges from about 0.001 to 30 mg/kg body weight, from about 0.01to 25 mg/kg body weight, from about 0.1 to 20 mg/kg body weight, or fromabout 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6mg/kg body weight. The skilled artisan will appreciate that certainfactors may influence the dosage required to effectively treat asubject, including but not limited to the severity of the disease ordisorder, previous treatments, the general health and/or age of thesubject, and other diseases present. Moreover, treatment of a subjectwith a therapeutically effective amount of an inhibitor can include asingle treatment or, in certain exemplary embodiments, can include aseries of treatments. It will also be appreciated that the effectivedosage of inhibitor used for treatment may increase or decrease over thecourse of a particular treatment. Changes in dosage may result from theresults of diagnostic assays as described herein. The pharmaceuticalcompositions can be included in a container, pack, or dispenser togetherwith instructions for administration.

It is to be understood that the embodiments of the present inventionwhich have been described are merely illustrative of some of theapplications of the principles of the present invention. Numerousmodifications may be made by those skilled in the art based upon theteachings presented herein without departing from the true spirit andscope of the invention. The contents of all references, patents andpublished patent applications cited throughout this application arehereby incorporated by reference in their entirety for all purposes.

The following examples are set forth as being representative of thepresent invention. These examples are not to be construed as limitingthe scope of the invention as these and other equivalent embodimentswill be apparent in view of the present disclosure, figures, table, andaccompanying claims.

Example 1 Stable Conformations of HIV Envelope Glycoprotein

Gp140 Trimer

Gp140, the ectodomain of the precursor gp160, is often produced to mimicthe prefusion state of the envelope, based on structural studies ofrelated other viral fusion proteins, such as, e.g., influenzahemagglutinin (Skehel and Wiley D C (2000) Ann. Rev. of Biochem. 69).However, the stability of HIV-1 gp140 varies greatly from strain tostrain and can be enhanced by adding a C-terminal trimerization tag suchas the T4-fibritin “foldon” or the coiled-coil trimer derived from GCN4(Yang et al. (2002) J. Virol. 76:4634). Recombinant SIV gp140 is astable trimer even without such a tag (Chen et al. (2000) J. Biol. Chem.275:34946). Gp140 proteins from a number of HIV-1 primary isolates wereexpressed with and without trimer tags to identify sequences that yieldparticularly stable gp140 trimers (FIG. 5). The construct 92UG-gp140-Fdwas derived from isolate 92UG037.8 and stabilized by a C-terminal foldontag. This construct proved to be especially well behaved (FIG. 1). Itsproperties, analyzed by size-exclusion chromatography, sedimentationequilibrium and chemical cross-linking, are shown in FIG. 2. Uncleavedgp140 from the same isolate but without the C-terminal foldon alsoyielded a stable trimer, but the foldon form was easier to purifybecause of its higher affinity for Ni-NTA. To mimic even more closelythe conformation of (gp120/gp41)₃ on the virion surface, partiallycleaved gp140 was generated using human plasmin (FIG. 6I and (Binley etal. (2002) J. Virol. 76:2606)), as recombinant furin is very ineffectivein vitro.

Gp41-Prehairpin Intermediate

To biochemically produce homogeneous forms of additional conformations,two constructs were designed to capture gp41 in the extended, prehairpinintermediate conformation. As shown in FIG. 1, gp41-inter has thefollowing sequence: (HR2)-linker-[HR1-CC loop-HR2-MPER]-(trimerizationtag), where HR1 and HR2 are the first and second heptad repeat motifsfound in gp41 from wild type HIV (the segments that form helices in thepostfusion trimer of hairpins) and the sequence in brackets isessentially the complete gp41 ectodomain, except for the fusion peptide.The “linker” is a short, flexible connector of serines and glycines.Without intending to be bound by scientific theory, when gp41-interchains trimerize, it was expected that the N-terminal HR2 segments wouldform a six-helix bundle with the HR1 segments, and the C-terminal HR2segments, constrained by the trimerization tag, would be unable to doso. The conformation of this construct could be pictured as theprehairpin intermediate captured by an HR2 peptide, such as T-20.Gp41-inter was expressed using sequences from two isolates: 92UG037.8and HXB2, with foldon and trimeric GCN4, respectively. In both cases,the protein could be expressed in E. coli and refolded in vitro. Controlexperiments showed that the N-terminal HR2 segment was required forrefolding of bacterially expressed protein as well as for obtainingsoluble, secreted protein from insect cells. A similar construct withthe gp41 sequence of SIVmac32H and the catalytic subunit of E. coliaspartate transcarbamoylase as a trimer tag (Chen et al. (2004) J.Virol. 78:4508) could likewise be obtained as secreted protein frominsect cells, indicating that the overall design is robust andindependent of the choice of a C-terminal trimerizing element.

Purified 92UG-gp41-inter was a monodisperse trimer (FIGS. 2D, E), stableeven after multiple rounds of gel-filtration chromatography. Its CDspectrum indicated a mixture of secondary structures (FIG. 7A).Negative-stain electron microscopy showed rod-like particles, 150 Å inlength and about 45 Å wide (FIG. 2F). The expected lengths for theN-terminal six-helix bundle and the C-terminal foldon are 75 and 28 Å,respectively. Without intending to be bound by scientific theory, theintervening segment of about 100 residues (C-C loop, HR2, and MPER) musthave a relatively compact fold, to span just 45 to 50 Å of axialdistance. The dry volume of three fully compact chains of this sizewould be about 35,000 Å³; the volume of an enclosing cylinder, 45 Å indiameter and 50 Å long, is about 75,000 Å³, compatible with normallevels of hydration for a folded protein.

Gp41 Postfusion Six-Helix Bundle

Forms of postfusion gp41 that contained the complete MPER tended toaggregate. A six-helix bundle construct that contained the full 2F5epitope (LDKWANL) (SEQ ID NO:14), but lacking the 4E10 epitope wasprepared (i.e., “gp41-post” (FIG. 1)). As refolded from E.coli-expressed inclusion bodies, gp41-post had all the propertiesexpected for a trimer of α-helical hairpins (FIG. 8).

Example 2 Ligand Binding and Antigenic Properties of Envelope Protein inDistinct Conformational States

A series of binding experiments were performed to verify the integrityof the 92UG-gp140-Fd trimer and to analyze its antigenic properties. The92UG-gp140-Fd trimer bound CD4 with a K_(d) of 1.98 nM (Table 1 and FIG.6A); the surface-plasmon-resonance (SPR) sensorgrams were fit using atwo-step binding model as used for binding of CD4 to gp120 core(Rits-Volloch et al. (2006) Embo J. 25:5026). Thus, the covalent linkagebetween gp120 and gp41 did not impede the conformational change in gp120that accompanies CD4 binding. The 92UG-gp140-Fd trimer also bound mAb2G12 (Trkola et al. (1996) J. Virol. 70:1100) with high affinity (Table1 and FIG. 6B) as expected, since the 92UG037 isolate was sensitive toneutralization by 2G12 (Binley et al. (2004) J. Virol. 78:13232). Thetrimer failed to bind the b12 IgG (Burton et al. (1994) Science266:1024), consistent with the resistance of the isolate toneutralization by that mAb (Binley et al. (2004) J. Virol. 78:13232),but the monomeric gp120 derived from 92UG037 did bind b12, with a K_(d)of 1.4 μM (Rits-Volloch et al. (2006) Embo J. 25:5026; and FIG. 6C).This affinity is about two orders of magnitude weaker than measured forthe same mAb with gp120 from isolates HXB2 or YU2 (Zhou et al. (2007)Nature 445:732). Without intending to be bound by scientific theory,this is likely due to a sequence difference in the CD4 binding loop(P369L (using HXB2 numbering), a residue that makes direct contact withb12 (Id.)). In addition, the 92UG-gp140-Fd trimer did not bind two othernon-neutralizing CD4 binding site antibodies, b6 and 15e, despite of thehigh affinities of these two antibodies to 92UG-gp120 core (Table 1;FIGS. 6D and 6E). Without intending to be bound by scientific theory, itwas concluded that the position and orientation of gp120 in theprefusion trimer reduced accessibility of the CD4 site to antibodieswithout impeding accessibility to CD4. Uncleaved 92UG-gp140-Fd alsobound a CD41 (CD4-induced) mAb, 17b (Thali et al. (1993) J. Virol.67:3978), but only in the presence of CD4, as expected (FIG. 6F). Thisresult indicated that the gp120 part of this trimer could undergo theconformational transition associated with formation of the bridgingsheet, the docking site for mAb 17b (and for co-receptor), even whengp120 could not fully dissociate, consistent with the similarobservations from other groups (Yang et al. (2002) J. Virol. 76:4634).

TABLE 1 ka (1/Ms) kd (1/s) Immobilized Flowing ka1 ka2 kd1 kd ligandanalyte (1/Ms) (1/s) (1/s) (1/s) Kd (M) 92UGgp140-Fd 4D sCD4 2.39E52.11E−2 1.41E−2 7.38E−4 1.98E−9 92UGgp140-Fd 2G12 Fab 5.35E4 9.58E−41.79E−8 b12 IgG 92UG gp120 1.50E4 1.49E−2 9.93E−7^(a) b6 IgG 92UG gp1205.62E4 1.51E−4 2.69E−9^(a) 15e IgG 92UG gp120 4.85E4 3.18E−3 6.57E−8^(a)2F5 Fab 92UGgp41- 1.00E4  1.39E−5^(b) 1.38E−9 inter-Fd 2F5 Fab HXB2gp41-6.82E3  6.11E−5^(b) 8.96E−9 inter-GCN4 92UGgp41-inter-Fd 2F5 Fab 1.53E54.96E−4 3.24E−9 2F5 Fab T20 4.54E5 2.17E−3 4.79E−9 2F5 Fab 2F5 epitope8.59E5 3.29E−3 3.83E−9 peptide^(c) 2F5 epitope 2F5 Fab 2.82E5 1.50E−35.33E−9 peptide^(c) 92UGgp41-inter-Fd 4e10scFv 1.60E5 1.71E−41.07E−9^(a) His-4e10scFv HXB2gp41- 1.49e5 4.43e−4 2.98e−9^(a)interf-GCN4 4e10 epitope 4e10scFv 3.69E4 6.85E−4 1.86E−8 peptide^(d) 2F5Fab 92UGgp41- 1.66E3 2.43E−3 1.41E−6 post

Table 1 depicts binding rate constants derived from SPR analysis.^(a)These binding constants were derived by fitting the sensorgram witha single concentration of analyte. The results presented here for b12IgG are essentially identical to those published previously by fittingruns with multiple concentrations (Chen et al. (2004) J. Virol.78:4508). ^(b)These sensorgrams are virtually flat during thedissociation phase, making accurate fitting very difficult. Thus,without intending to be bound by scientific theory, the actual off-ratesare probably even slower than those listed here. ^(c)The short 2F5epitope peptide used was ELLELDKWASL (SEQ ID NO:15). ^(d)The 4E10epitope peptide was biotin-SLWNWFNITNWLWYIK (SEQ ID NO:16) (Alam et al.(2007) J. Immunol. 178:4424)

Antibodies to gp41 include those in “cluster I,” which bind theimmunodominant, disulfide-containing loop between the two helicalregions of the postfusion form, and those in “cluster II,” which bindMPER epitopes. The 92UG-gp140-Fd trimer bound tightly with two cluster ImAbs, 240-D and 246-D (FIG. 6G), in accord with earlier observationsthat SIV gp140 trimer interacts with cluster I mAbs, KK41 and 9G3, andthat the cluster I epitopes are well-exposed on the primary HIV-1 nativevirions (Xu et al. (1991) J. Virol. 65:4832; Kim et al. (2001) J. Biol.Chem. 276:42667; Nyambi et al. (2000) J. Virol. 74:7096). We note thatmAb 9G3 has neutralizing activity (Kim et al. (2001) J. Biol. Chem.276:42667). We also find strong binding of 240-D and 246-D withplasmin-cleaved 92UG-gp140-Fd, but as the cleavage is incomplete, onecannot make strong conclusions about the effects of gp120-gp41 cleavageon antibody affinity. Some previous reports suggest that cluster I andII epitopes are exposed on uncleaved, oligomeric gp140 but inaccessibleon cleaved, disulfide-linked, monomeric SOS gp140 derived from the samestrain (Schulke et al. (2002) J. Virol. 76:7760). The conformationalhomogeneity of those preparations was not fully assessed, and thecluster I epitope in the SOS gp140 was also altered by the extradisulfide introduced.

The 92UG037 isolate was sensitive to neutralization by the broadlyneutralizing, MPER-directed human monoclonal antibodies, 2F5 and 4E10(Binley et al. (2004) J. Virol. 78:13232), and these two antibodiesindeed recognized unfolded 92UG-gp140-Fd on a western blot (FIG. 3A,inset, lane 1; FIG. 4C). The native trimer did not, however, bind mAb2F5 under any of the conditions that were tested by SPR. In particular,92UG-gp140-Fd exhibited no interaction with a 2F5 surface, regardless ofwhether IgG or Fab was used for immobilization (FIG. 3A). Also, nobinding of 2F5 IgG or Fab with immobilized gp140, at either 20° or 37°C., was observed. The presence or absence of the foldon tag had noeffect, nor did partial plasmin cleavage (FIG. 3A). The 92-gp140-Fdtrimer also failed to bind 4E10 Fab and showed only very weak binding ifany at all to 4E10 IgG at high concentration (FIG. 4A and FIG. S2H).Without intending to be bound by scientific theory, it was concludedthat the epitopes of 2F5 and 4E10 were either buried or in anon-antigenic configuration on the native gp140 trimer. This conclusionis consistent with published reports that mAb 2F5 does not bind theenvelope protein on the surface of virions (Cavacini et al. (2002) AIDS16:2409; Hart et al. (2003) J. Gen. Virol. 84:353). Other publishedexperiments suggest a temperature-sensitive interaction withcell-surface expressed Env, but the structural heterogeneity of cleavedenvelope protein on cell surfaces and the potential lipid-bindingability of 2F5 make those results difficult to interpret (Finnegan etal. (2002) J. Virol. 76:12123; Sattentau et al. (1995) Virology 206:713;Haynes et al. (2005) Science 308:1906). Those gp140 preparationsreported to bind 2F5 all contain significant amounts of monomers,dimers, or aggregates (Schulke et al. (2002) J. Virol. 76:7760; Jeffs etal. (2004) Vaccine 22:1032; Dey et al. (2007) Virology 360:199).

If mAbs 2F5 and 4E10 do not bind the ectodomain of the (gp120/gp41)₃ inthe prefusion conformation found on virions, how do they neutralize?Previous attempts to mimic the intermediate state have been limitedlargely to constructs containing only the HR1 and HR2 fragments (Eckertet al. (1999) Cell 99:103; Root et al. (2001) Science 291:884; Binley etal. (2003) J. Virol. 77:5678; de Rosny et al. (2004) J. Virol. 78:2627),which could not be used to resolve the issue. In contrast, thegp41-inter constructs described herein contained nearly the full-lengthgp41 ectodomain, including the full epitopes for 2F5 and 4E10. The datain FIGS. 3B and 7B show that the Fab fragment derived from mAb 2F5 boundgp41-inter proteins very tightly (K_(d)<10 nM, with an off rate slowerthan 1.4×10⁻⁵ s⁻¹), regardless of the choice of isolate andtrimerization tag (Table 1) (The Fab was used to avoid potential avidityeffects with intact antibody). The estimated dissociation constant wasrelatively insensitive to which protein was immobilized on the chip. Thecomplex of 2F5 Fab and 92UGgp41-inter protein could also be purified bygel-filtration chromatography. The 4E10 single-chain Fv fragment (scFv)likewise showed very strong binding to gp41-inter proteins(K_(d)˜1.1-2.9 nM; FIGS. 4B and 7D; Table 1). ScFv was used because 4E10Fab produced by papain-digestion showed weaker neutralizing activitythan those of scFv and IgG, while the latter two were equally potent.These observations indicate that 2F5 and 4E10 exerted their neutralizingactivity by binding an intermediate conformation of gp41. Kineticstudies of membrane fusion have shown that both 2F5 and 4E10, like T20,are probably effective only during a small time interval during thefusion process (Binley et al. (2003) J. Virol. 77:5678; Dimitrov et al.(2007) Biochemistry 46:1398). Moreover, mutations in the gp41 core thatdestabilize the six-helix bundle conformation enhance sensitivity of themutant viruses to 2F5 neutralization (Follis et al. (2002) J. Virol.76:7356).

Peptides that contained the 2F5 epitope, such as T-20, bound the 2F5Fab, as expected (FIG. 3D and Table 1), but they dissociated much morerapidly than did the gp41-inter proteins (Table 1). A rapid off rate hasalso been reported when the 2F5 epitope peptide is inserted into proteinscaffolds other than gp41-inter (Ho et al. (2005) Vaccine 23:1559).Likewise, a peptide containing the full 4E10 epitope also showed weakerbinding to 4E10 scFv (K_(d)˜18 nM) than did gp41-inter (FIG. 4D andTable 1). Thus, without intending to be bound by scientific theory, verystrong binding by these two mAbs appears to be a specific consequence ofincorporating the epitope into a prehairpin intermediate conformation.

As expected, the postfusion state of gp41 bound the 2F5 Fab very weakly(K_(d)˜1.4 μM; Table 1; FIG. 7C), while a short epitope peptide endingwith the same residue as gp41-post showed much tighter binding(K_(d)˜3.8-5.3 nM; Table 1; FIG. 8D), indicating that the 2F5 epitope ingp41-post did not have an optimal binding conformation. This result isconsistent with observations that the formation of the six-helix bundleweakens 2F5 binding (Gorny and Zolla-Pazner (2000) J. Virol. 74:6186).

Example 3 Discussion

The results presented herein indicate that 2F5 and 4E10 inhibit HIV-1infection by binding to their epitopes as displayed on the prehairpinintermediate conformation of gp41, thereby blocking a crucial step inthe conformational transition required for membrane fusion. Binding maynot obstruct formation of the six-helix bundle, however, as the epitopeslie outside HR2. That is, the block may occur at a very late step in the“zipping up” of gp41. Without intending to be bound by scientifictheory, these two antibodies could, for example, prevent MPER frominteracting with residues proximal to the fusion peptide, a potentiallyrequired step for induction of membrane hemifusion. Thenon-neutralizing, cluster I antibodies bound gp41-inter as well asprefusion gp 140 (FIGS. 6G and 7C). Thus, binding to the intermediateconformation is not by itself sufficient for neutralization. Becausetheir target is a transient intermediate, 2F5 and 4E10 have a relativelynarrow “window of opportunity.” Both antibodies, which recognize linearepitopes adjacent to each other in the MPER of gp41, have long,hydrophobic heavy-chain CDR3 loops. These loops contact bound MPERpeptides only at their base, and it has been proposed that they alsointeract with the viral membrane (Ofek et al. (2004) J. Virol. 78:10724;Cardoso et al. (2005) Immunity 22:163). Without intending to be bound byscientific theory, the putative, relatively non-specific membranebinding may simply concentrate the antibody to give it a kinetic headstart during the short lifetime of the intermediate. Indeed, both 2F5and 4E10 Fab fragments bind gp41-inter with high affinity in the absenceof a lipid bilayer, consistent with a largely kinetic role for anymembrane interaction.

Haynes and colleagues have found that 2F5 and 4E10 have propertiesresembling those of autoreactive antibodies (including their long,heavy-chain CDR3 loops) and that they interact with phospholipids(Haynes et al. (2005) Science 308:1906). They suggest that thesecharacteristics might lead to elimination of such heavy chains from theavailable repertoire, increasing the challenge of making immunogens toelicit MPER-reactive responses. The data presented herein provides anadditional explanation for the rarity of 2F5-like antibodies in HIVinfected individuals. The estimated exposure time for a T-20 target siteduring cell-cell fusion is about 15 minutes (Muñoz-Barroso et al. (1998)J. Cell Biol. 140, 315-323). Recent estimates for the lifetime of anintermediate sensitive to the construct known as “5-helix” (asingle-chain model for five of the six helices in the postfusion bundle)are much lower, on the order of only 5-10 seconds (Steger and Root(2006) J. Biol. Chem. 281:25813). In either case, the transientconformation would not have a long enough lifetime to be effective ininducing a host response. Moreover, it would be present only at theinterface of an infecting virion with a T-cell or macrophage,inaccessible to the B-cell receptor that must initiate clonalproliferation and antibody synthesis.

Various examples from other viruses illustrate that the relevantconformation of a viral envelope protein must be presented, if immunogendesign is the goal. The exposure of flavivirus neutralizing epitopesdepends on whether the E protein is in a pre- or post-fusionconformation (Modis et al. (2004) Nature 427:313). A similar conclusionfollows from the mapping of antigenic sites on the surfaces of pre- andpost-fusion vesicular stomatitis virus (VSV) glycoprotein G (Roche etal. (2007) Science 315:843) and from early studies on antigenicity ofinfluenza virus (Daniels et al. (1983) J. Gen. Virol. 64 (Pt 8):1657).HIV-1 Env-based protein immunogens often induce high ELISA-titerantibody responses with limited neutralizing activity and breadth (Boweret al. (2006) Vaccine 24:5442), but lack of rigorously characterizedpreparations of the envelope proteins in well-defined conformationalstates has confused many analyses of antigenicity and immunogenicity.Preparations of recombinant gp140 are often mixtures of monomers andhigher oligomers. Their conformation and physiological relevance aredifficult to define. Even cell-associated or virion-associated envelopeproteins are structurally heterogeneous because of the tendency forgp120 to dissociate and because of inefficient cleavage of theprecursor. Accordingly, the preparations described herein are a usefulstandard against which to evaluate future immunogens. The tight bindingof 2F5 to gp41-inter provides evidence for the significance of anextended, prehairpin intermediate in the fusion transition. Moreover,gp41-inter may provide a scaffold for presenting the MPER in aconformation relevant to neutralization and potentially for inducing arelevant B-cell response.

Example 4 Materials and Methods

Expression Constructs

Expression constructs were generated by standard PCR techniques.Constructs for fusion proteins were made either by overlapping PCR or byligation of compatible restriction fragments. pET21-a(+) and pET23-a(+)(Novagen, La Jolla, Calif.) were used for expression of 92UG-gp41-inter,and of HXB2-gp41-inter, 92UG-gp41-post, respectively, in E. coli.PFastBac-1 (Invitrogen, Carlsbad, Calif.) was the expression vector forgp140, gp140-Fd constructs in insect cells. P92UG-gp140 containsresidues 26-675 (92UG037.8 numbering), followed by four residuesintroduced by restriction sites EcoR I and XbaI, a factor Xa site and aHis-tag (-EFSRIEGRHHHHHH (SEQ ID NO:7)). P92UG-gp140-Fd includesresidues 26-675, followed by four residues introduced by restrictionsites EcoRI and XbaI, a factor Xa site, the foldon tag and a His-tag(EFSRIEGRGSGGYIPEAPRDGQAYVRKDGEWVLLSTFLGHHHHHH (SEQ ID NO:8)).P92UG-gp41-inter-Fd begins with residues 612-657 (HR2) and a linker(GGSGG (SEQ ID NO:9)), followed by residues 531-675, the foldon tag anda His-tag (GTGGSGGYIPEAPRDGQAYVRKDGEWVLLSTFLGHHHHHH (SEQ ID NO:10)). Dueto the initial confusion about the Env clone used, the primers for PCRreactions were designed based on the sequence of another 92UG037 isolate(GenBank No. U51190). As a result, following changes were introduced inthe p92UG-gp41-inter-Fd: residues 533 (V to A), 612 (R to D), 651 (E toD) and 689 (N to K). P92UG-gp41-post contains residues 526-573, a linker(SGGRGG (SEQ ID NO:11)) and residues 620-660. PHXB2-gp41-inter-GCN4begins with residues 620-661 (HR2, HXB2 numbering), a linker (SGGRGG(SEQ ID NO:12)), followed by residues 546-678, and finally the GCN4trimerization domain (IEDKIEEILSKIYHIENEIARIKKLIGE (SEQ ID NO:13)).PHXB2-gp41-interf-GCN4 is essentially identical as pHXB2-gp41-inter-GCN4except the C-terminal end of gp41 ectodomain was extend to residue 683to include the full epitope for mAb 4E10. The constructs were verifiedby restriction digestion and DNA sequencing.

Expression in E. coli and Protein Refolding

Gp41-inter and gp41-post constructs were expressed in E. coli usingRosetta (DE3)pLysS cells, which supply tRNAs for rare codons. Bacterialcultures were induced at an OD600 of 1.0 by addition of 1 mMisopropyl-D-thio galactopyranoside (IPTG). Cells were harvested 2-3hours post-induction by centrifugation. Cell pellets were frozen at −80°C. Env constructs were insoluble when expressed in E. coli. Cells werelysed by three cycles of freezing-thaw in PBS with 0.4 mg/ml DNase I,0.4 mg/ml RNase A, 2 mg/ml lysozyme, followed by brief sonication. Forhis-tagged constructs, inclusion bodies were spun down bycentrifugation, and solubilized in 6 M guanidine hydrochloride (GdnHCl).After removing insoluble materials, the supernatant was loaded onto aNi-NTA resin, washed with 6M GdnHCl, eluted with 300 mM imidazole in 6 MGdnHCl. The fractions containing His-tagged protein were pooled and theprotein refolding initiated by rapid dilution of the pooled fractionsinto ice-cold refolding buffer (1 M arginine, 100 mM Tris-HCl pH 7.5, 2mM EDTA, 0.2 mM oxidized glutathione, 2 mM reduced glutathione, oneprotease cocktail tablet (Roche, Basel, Switzerland)) at a final proteinconcentration of 100 μg/ml. The refolding mix was stored at 4° C. for atleast 24 hours. The refolding reaction was then dialyzed against PBSfour times and purified by a Ni-NTA column under native conditions. Theimidazole-eluted fractions were pooled, concentrated, and furtherseparated away from aggregated species by gel-filtration chromatographyon Superdex 200 (GE Healthcare, United Kingdom) with a buffer containing25 mM Tris-HCl (pH 7.5) and 150 mM NaCl. The purified protein wasconcentrated and stored at −80° C. Non-his-tagged gp41 constructs waspurified be an acid-extraction method described in Frey et al. ((2006)Proc. Natl. Acad. Sci. USA 103:13938). The protein refolding proceededthe same way described above, except that refolding mix after dialysiswas concentrated by ultrafiltration using Centriconplus-70 (Millipore).

Expression in Insect Cells and Protein Purification

The gp140 proteins were expressed in insect cells using the Bac-to-Bacsystem (Invitrogen) as described (Chen et al. (2000) J. Biol. Chem.275:34946). Briefly, recombinant baculovirus was generated according tothe manufacturer's protocol and amplified in Sf9 insect cells. Theoptimal amount of virus and post-infection harvest time was determinedby small-scale tests in 6-well plates. For large-scale production, 12 Lof Sf9 or T. ni (Hi-5) cells (2×10⁶ cells/ml) were infected at theoptimal MOI. The supernatant was harvested 72 hours post-infection bycentrifugation and concentrated to 2 L in a tangential flow filtrationsystem, ProFlux M12 (Millipore), followed by immediately exchanging intoPBS in the ProFlux M12. After a clarifying spin and adding imidazole tothe final concentration of 15 mM, the supernatant was loaded onto anickel column at a flow rate of 1 ml/min, then washed with 15 mMimidazole in PBS, followed by further washing with 40 mM imidazole inPBS. The protein was eluted with 300 mM imidazole in PBS. The fractionscontaining the purified protein were pooled, concentrated, and furtherpurified by gel filtration chromatography on Superose 6 (GE Healthcare).The protein was concentrated, frozen in liquid nitrogen and stored at−80° C.

Production of Monoclonal Antibody and Fab Fragments

Monoclonal antibodies were purified from cell supernatants of hybridomasgrowing in roller bottles using a 5 ml GammaBind Plus Sepharose affinitycolumn as described previously (Chen et al. (2000) J. Biol. Chem.275:34946). The 2G12 Fab was kindly provided by Robyn Stanfield, IanWilson and Dennis Burton at Scripps. 2F5 Fab fragment was produced bydigestion of IgG with Endoproteinase Lys-C (Roche) as published (Ofek etal. (2004) J. Virol. 78:10724). 4E10 Fab was generated using a protocolmodified from Cardoso et al. ((2005) Immunity 22:163). Briefly, 4E10 IgGwas digested by activated papain (Sigma) in 20 mM sodium phosphate, pH7.0 and 10 mM EDTA. The digest was then dialyzed against 0.1 M sodiumacetate, pH 5.5 overnight. The Fab was purified on a protein A columnusing buffers supplied in ImmunoPure Fab Preparation Kit (Pierce). Theflow-through from the protein A column was concentrated and furtherpurified by a Superdex 200 column using 200 mM sodium acetate, pH 5.5 asa running buffer. All the purified Fabs were analyzed by SDS-PAGE underboth reducing and non-reducing conditions. Production andcharacterization of 4E10 single chain Fv fragment was performed.Briefly, 4E10 scFv with a C-terminal His-tag was cloned into pET-21a(+)vector and expressed in E. coli. The protein was refolded and purifiedfollowing the same protocol for 92UGgp41-inter-Fd (see Expression in E.coli and protein refolding).

Chemical Crosslinking, Analytical Ultracentrifugation (AUC) and CircularDichroism Spectroscopy

Chemical crosslinking, analytical ultracentrifugation (AUC) and circulardichroism spectroscopy were carried out as described previously (Chen etal. (2000) J. Biol. Chem. 275:34946).

Surface Plasmon Resonance Binding Assays

All experiments were performed in duplicate with a Biacore 3000instrument (Biacore Inc.) at 20° C. in HBS-EP running buffer (10 mMHEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% surfactant P20).Immobilization of ligands to CM5, NTA and SA chips (Biacore Inc.)followed the standard procedures recommended by manufacture.Immobilization of 4E10 antibody to a CM5 chip by the standard aminecoupling procedure was found to block binding to its antigens and thesame protocol to couple 92UGgp140-Fd trimer to a CM5 chip also has adenaturing effect on the trimer. These two types of immobilization weretherefore not used in the subsequent experiments. The finalimmobilization levels were between 300 and 500 RU to avoid rebindingevents. For kinetic measurements, sensorgrams were obtained by passingvarious concentrations of an analyte over the ligand surface at a flowrate of 50 μl/min using a 2 minute association phase and a 10 minutedissociation phase. The sensor surface was regenerated between eachexperiment using a single injection of 35 mM NaOH, 1.3 M NaCl; or 10 mMHCl, 1.3 M NaCl, at a flow rate of 100 μl/minute. Identical injectionsover blank surfaces were subtracted from the data for kinetic analysis.Binding kinetics was evaluated using BiaEvaluation software (BiacoreInc.).

Negative-Stain Electron Microscopy

92UGgp41-inter protein was negatively stained with uranyl formate asdescribed (Ohi et al. (2004) Biol. Proceed. Online 6:23). Images wererecorded by a Tecnai T20 microscope operated at 120 kV with amagnification of 50K, and by a Gatan 4K×4K CCD camera with a defocus of1.5 μm following a low-dose procedure. All images were binned 2×2 to afinal pixel size of 4.5 Å/pixel at specimen level. Individual particleswere selected manually and processed with SPIDER (Frank et al. (1996) J.Struct. Biol. 116:190).

What is claimed:
 1. An isolated, antigenic human immunodeficiency virustype 1 (HIV-1) gp41 fusion polypeptide capable of forming a prehairpinintermediate conformation comprising the following structure: NH₂-heptadrepeat 2 (HR2)-linker-heptad repeat 1 (HR1)-C-C-immunodominant loopregion-HR2-membrane proximal external region (MPER)-COOH, wherein saidfusion peptide is capable of inducing a broadly anti-HIV-1 neutralizingantibody when injected into a subject.
 2. The polypeptide of claim 1,further comprising an oligomerization domain carboxy terminal to themembrane-proximal external region.
 3. The polypeptide of claim 2,wherein the oligomerization domain is a trimerization domain.
 4. Thepolypeptide of claim 1, further comprising a protein tag carboxyterminal to the membrane-proximal external region.
 5. The polypeptide ofclaim 1, wherein the polypeptide elicits production of a broadlyneutralizing antibody when injected into a subject.
 6. An immunogeniccomposition comprising an isolated antigenic human immunodeficiencyvirus type 1 (HIV-1) gp41 fusion polypeptide capable of forming aprehairpin intermediate conformation comprising the following structure:NH₂-heptad repeat 2 (HR2)-linker-heptad repeat 1(HR1)-C-C-immunodominant loop region-HR2-membrane proximal externalregion (MPER)-COOH, and a pharmaceutically acceptable carrier.